Cartridge-based thermocycler

ABSTRACT

Cartridge-based thermocyclers can include cartridges that are configured to move a fluid between distinct chambers. In some cases, the cartridge-based thermocyclers can be used for thermocycling a sample fluid comprising a deoxyribonucleic acid (DNA) target to perform polymerase chain reaction (PCR). Individual chambers can be heated, cooled, and/or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber. The cartridges can have a laminate construction. The cartridges can be configured to enable multiplexed thermocycling and/or detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/228,709, filed Dec. 20, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/124,334, filed Sep. 7, 2016 (now U.S. Pat. No.10,195,610), which is national stage entry of International PatentApplication No. PCT/US2015/019497, filed Mar. 9, 2015, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/950,769,filed Mar. 10, 2014, which applications are incorporated herein byreference.

SUMMARY

The present disclosure provides systems for performing thermal cycling(also “thermocycling” herein) of a fluid (also “sample” or “samplefluid” herein). The systems herein can be used for thermocycling asample fluid to perform biological or biochemical analysis. In someexamples, the systems herein can be used for thermocycling a samplefluid comprising a deoxyribonucleic acid (DNA) target to performpolymerase chain reaction (PCR). The systems can be cartridge-basedsystems (also “cartridges,” “cartridge-based thermocyclers” or“cassettes” herein) that enable low-cost, disposable PCR systems to berealized. In some cases, the cartridges can be real-time, multiplexedsystems (also “multiplexed assays” herein). The cartridges can becoupled to other PCR system components, such as, for example, one ormore other cartridges and/or an instrument. In some implementations, PCRsystems comprising disposable cartridge system(s) coupled with a durableinstrument can be provided.

Systems of the present disclosure include cartridges that are configuredto move fluid between distinct chambers. Each chamber can have a giventemperature, composition, volume and/or shape. Individual chambers canbe heated, cooled, and/or compressed to mix fluid within the chamber orto propel fluid in the chamber into another chamber. Further, thechambers can be shaped to inhibit trapping of air bubbles. The chamberscan be configured to allow rapid thermal equilibration. In someimplementations, the cartridge comprising the chambers can have alaminate construction.

The present disclosure relates to a thermocycler comprising a firstchamber for holding a fluid at a first average temperature and a secondchamber for holding the fluid at a second average temperature. Thesecond chamber is in fluid communication with the first chamber, whereinthe fluid is transferred between the first chamber and the secondchamber to achieve a transition from the first average temperature tosubstantially the second average temperature or vice versa at a rate of10 μL° C./second or more. The first and second chambers can be providedon a disposable portion of the thermocycler. In some cases, thetransition from the first average temperature to substantially thesecond average temperature or vice versa can be achieved at a rate of 25μL° C./second or more. The thermocycler can have a cycle time of 10seconds or less. The fluid can have a starting volume of about 25 μL ormore. In one embodiment, the first average temperature is nominallybetween about 55° C. (328 K) and about 65° C. (338 K), and the secondaverage temperature is nominally about 95° C. (368 K). In oneembodiment, the fluid has a starting volume of about 25 μL or more, forexample, about μL. In one embodiment, the fluid is transferred betweenthe first chamber and the second chamber to achieve the transition fromthe first average temperature to substantially the second averagetemperature or vice versa within 5 seconds or less, 4 seconds or less, 3seconds or less, 2 seconds or less, or 1 second or less. In oneembodiment, the thermocycler comprises a filling and/or venting channel,wherein the venting channel prevents gases from being trapped duringfilling.

The present disclosure is directed to a method for performing polymerasechain reaction (PCR) comprising providing a first fluid holding chamberhaving a first average temperature, providing a second fluid holdingchamber having a second average temperature, mechanically actuatingfluid transfer between the first chamber and the second chamber, andcompleting the PCR within a total thermocycling time that is at leastabout 9 times shorter than a corresponding thermocycling time on aconventional system. The method can further comprise completing the PCRamplification within a total thermocycling time of less than about 4minutes. The method can further comprise completing the PCR within atotal thermocycling time that is at least about 11.5 times shorter thanthe corresponding thermocycling time on a conventional system. Themethod can further comprise completing the PCR at a PCR efficiency thatis substantially the same as a PCR efficiency of the conventionalsystem. The PCR efficiency can be at least about 92%. In one embodiment,the method further comprises detecting PCR amplification by monitoringthe first chamber, the second chamber or a channel between the firstchamber and the second chamber. Monitoring includes opticalmultiplexing. In one embodiment, a PCR amplification method is completedin a time that is about 11.5 times shorter than the correspondingthermocycling time on a conventional system. In one instance, the PCRefficiency is substantially the same as a PCR efficiency of theconventional system. In another instance, the PCR amplification is equalto an amplification of the conventional system. In one embodiment, thePCR method is complete upon reaching a predetermined number of cycles.

The present disclosure provides a low-cost polymerase chain reaction(PCR) system comprising a cartridge configured for transferring a fluidbetween a first chamber and a second chamber maintained at distincttemperatures, wherein the cartridge has a laminate construction definingthe first chamber and the second chamber, and wherein the transfer ofthe fluid between the first chamber and the second chamber is forthermocycling the fluid. The laminate construction can define the firstchamber and the second chamber in the absence of mechanical force ormechanical actuation. In one embodiment, the cartridge is disposable. Inone embodiment, the volumes of the first chamber and the second chamberdepend on the thickness of individual layers of the laminateconstruction. In one implementation, the laminate construction comprisesa first outer plastic layer, a first pressure sensitive adhesive layer,a second pressure sensitive adhesive layer, a second outer plasticlayer, and optionally a cover. In one embodiment, the cover is a rigidstructure. In one embodiment, the cover is bonded to the first outerplastic layer or the second outer plastic layer. In another embodiment,the first outer plastic layer or the second outer plastic layer is amembrane layer. In one embodiment, the starting volume of the fluid isat least about 25 μL, at least about 50 μL, and/or at least about 60 μL.In one embodiment, the height of the first chamber and the secondchamber is 250 μm or less. In one embodiment, the first chamber or thesecond chamber has a tear drop shape. In another embodiment, the firstchamber or the second chamber or both are shaped to achieve a reducednumber of nucleation sites.

In one embodiment, the low-cost polymerase chain reaction (PCR) systemhas a laminate construction comprising an optical window. In oneembodiment, the optical window provides an optical path to a portion ofa fluid path between the first chamber and the second chamber. In oneembodiment, a sample volume is interrogated through the optical window.In another embodiment, the optical window comprises a light directingfeature. A light directing feature includes, without limitation, a lens,prism, Fresnel lens or any combination thereof. In a further embodiment,the system further comprises a blocking feature to obstruct stray lightfrom an excitation source. Blocking features include, withoutlimitation, foil, coatings or a combination thereof.

The present disclosure further provides a multiplexed assay comprising aplurality of thermocycling units. Each thermocycling unit comprises afirst chamber for holding a fluid at a first average temperature. Eachthermocycling unit further comprises a second chamber for holding thefluid at a second average temperature. The second chamber is in fluidcommunication with the first chamber. The first chamber and the secondchamber each have a non-zero volume prior to holding the fluid. In oneembodiment, the assay further comprises a detector coupled to at least asubset of the plurality of thermocycling units. In one embodiment, thedetector is dedicated to a single thermocycling unit of the plurality ofthermocycling units. In one embodiment, the detector is shared by atleast a subset of the plurality of thermocycling units. In oneembodiment, at least a subset of the plurality of thermocycling unitsare identical. In another embodiment, a first subset of the plurality ofthermocycling units has a different configuration than a second subsetof the plurality of thermocycling units.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings or figures (also “FIG.” and “FIGs.” herein), ofwhich:

FIG. 1 shows a cross-sectional view of a thermocycling unit and a topview of fluid chambers on the thermocycling unit.

FIG. 2A shows temperature of fluid in right chamber (hot) immediatelyafter fluid transfer from left chamber (cold). The time to achieve thehot average temperature is only about 5 seconds. The temperature isexpressed in degrees Kelvin (K).

FIG. 2B shows a histogram of temperatures of the fluid entering the hotchamber in FIG. 2A. Most of the fluid is at the hot temperature ±3° C.(3 K) in about 5 seconds. The temperature is expressed in degrees Kelvin(K).

FIG. 3 shows a layered cartridge without separate optical window.Readout is directly in one of the wells.

FIG. 4 is an exploded view of separate layers of a layered cartridgethat includes an embedded optical window.

FIG. 5 shows front, back and side views of an assembled laminatedcartridge with optical window embedded.

FIG. 6 is an exploded view of a three-layer “blister pack” disposable.

FIG. 7 are cut-away and contour views of an assembled laminatedcartridge with an optical window embedded.

FIG. 8 shows a cartridge thermally modeled in Comsol assuming one-sidedor two-sided heating through 50 micron (50 μm) thick polyimide film(s).

FIG. 9A provides thermal modeling results for the cartridge in FIG. 8with two-sided heating, showing that temperatures are reached within 2seconds.

FIG. 9B shows a histogram of fluid temperatures entering the coldchamber (55° C.) from the hot chamber (95° C.), as modeled in FIG. 9A.

FIG. 10A provides thermal modeling results for the cartridge in FIG. 8with two-sided heating, showing that temperatures are reached within 2seconds.

FIG. 10B shows a histogram of fluid temperatures entering the hotchamber (95° C.) from the cold chamber (55° C.), as modeled in FIG. 10A.

FIG. 11A provides thermal modeling results for the cartridge in FIG. 8with one-sided heating, showing that temperatures are reached within 4seconds.

FIG. 11B shows a histogram of fluid temperatures entering the coldchamber (55° C.) from the hot chamber (95° C.), as modeled in FIG. 11A.

FIG. 12A provides thermal modeling results for the cartridge in FIG. 8with one-sided heating, showing that temperatures are reached within 4seconds.

FIG. 12B shows a histogram of fluid temperatures entering the hotchamber (95° C.) from the cold chamber (55° C.), as modeled in FIG. 12A.

FIG. 13 shows fluid contact angles of a 10 μL droplet on variousmaterial surfaces.

FIG. 14A provides a working embodiment of a laminated cartridge.

FIG. 14B illustrates a recorded demonstration of PCR in a laminatedstructure.

FIG. 15 shows a cartridge with optical components.

FIG. 16 shows an embodiment of a multichamber cartridge.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions occurs to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein areemployable. It shall be understood that different aspects of theinvention can be appreciated individually, collectively, or incombination with each other.

The term “polymerase chain reaction (PCR),” as used herein, generallyrefers to any variation on the basic process or operation of amplifyinga single or a few copies of a specific region of a DNA strand (alsoreferred to as “DNA target,” “target,” or “target DNA” herein) togenerate copies of a particular DNA sequence. In some examples, DNAfragments of between about 100 and 40,000 base pairs (bp) can beamplified. Primers (e.g., short DNA fragments) containing sequencescomplementary to the target region along with a DNA polymerase (e.g.,Taq polymerase or another heat-stable DNA polymerase) can be provided toenable selective and repeated amplification. As PCR progresses, the DNAgenerated can itself be used as a template for replication. Componentsneeded to perform PCR can include, but are not limited to, primers, DNApolymerase, DNA building blocks (e.g., deoxynucleoside triphosphatenucleotides), buffer solution, divalent cations (e.g., magnesium ormanganese ions), and monovalent cations (e.g., potassium ions). Thermalcycling (e.g., alternate heating and cooling of the PCR sample) througha defined series of temperature steps can be used. In some examples, aseries of 20-40 repeated temperature changes (also “cycles” herein) canbe used, with each cycle comprising two or three discrete temperaturesteps, as described in greater detail below. In some cases, the cyclingcan be preceded by and/or followed by additional temperature step(s).The temperatures used and the length of time they are applied in eachcycle can depend on a variety of parameters (e.g., enzyme used for DNAsynthesis, concentration of divalent ions and nucleotides in thereaction, melting temperature of the primers).

The term “amplification,” as used herein, generally refers to arelationship between an amplified target concentration and an initialtarget concentration. The amplification can be defined as, for example,A=C_(x)/C₀, where C_(x) is the amplified target concentration (e.g., DNAcopies per volume) and C₀ is the initial target concentration (e.g., DNAcopies per volume). In an example, an initial concentration of BacillusAtrophaeus (B. Atro) DNA of about C₀=10⁵ copies per milliliter (mL) isrun through 30 cycles of PCR thermocycling to achieve an amplifiedtarget concentration of about C_(x)=10¹³ copies/mL, and thus anamplification of about A=10⁸. At a PCR efficiency of 100%, each cyclecan double the target population, and at 30 cycles, an amplification ofabout 2³⁰ or about A=10⁹ can be achieved. Since the amplification inthis example is less than 10⁹, the PCR efficiency is less than 100%. Forexample, the PCR efficiency can be about 84% or about 92%. In someexamples, PCR can continue to a given amplification, where a targetpopulation (also “target concentration” herein) is easily observed(e.g., by observing an optical signal produced by a bound fluorescentprobe). In some examples, reaching a given number of cycles can be usedto define completion of the PCR (i.e., PCR completion can be defined asa point when a given number of cycles have been completed).Amplification includes, without limitation, nucleic acid amplification.Exemplary nucleic acid amplification reactions include, withoutlimitation, polymerase chain reaction (PCR), strand displacementamplification (SDA), helicase dependent amplification (HDA),loop-mediated isothermal amplification (LAMP), transcription-mediatedamplification (TMA), nucleic acid sequence based amplification (NASBA)and self-sustained sequence replication (3SR). Nucleic acidamplification reactions include both real-time and end-point reactions.

The term “cycle time,” as used herein, generally refers to the timerequired to accomplish a denaturing step, an annealing step, and anextension step in a nucleic acid amplification reaction. In an exemplaryembodiment, these steps are done at three distinct temperatures or twodistinct temperatures. In an example where these steps are accomplishedat two distinct temperatures in two respective cartridge chambers, thecycle time is the residence time of a sample in two consecutive chambers(e.g., a hot chamber and a cold chamber) of a cartridge.

The term “ramp time,” as used herein, generally refers to the timerequired for the bulk of a fluid to ramp from a first temperature to asecond temperature.

The term “extension time,” as used herein, generally refers to the timerequired for a DNA polymerase (e.g., Taq polymerase) to extend thelength of the copied molecule. A typical extension rate for Taqpolymerase can be 1000 nucleotides per second at 72° C. The extensiontime for a PCR product on the order of 200-400 bp using thecartridge-based thermocyclers of the present disclosure can be, forexample, 1-2 seconds.

The term “melt time,” as used herein, generally refers to the timerequired to achieve adequate melting of the DNA of the copied molecule(e.g., by disrupting hydrogen bonds between complementary bases,yielding single-stranded DNA molecules).

The term “dwell time,” as used herein, generally refers to the time thata sample resides at each temperature. For example, for two-temperaturePCR, cycle time can equal the dwell time times two. In some cases, cycletime can be a sum of a first dwell time at a first temperature and asecond dwell time at a second temperature. In an example, the dwell timeequals the residence time of a sample in an individual chamber (e.g., ahot chamber or a cold chamber) of a cartridge. In some cases, dwell timecan equal ramp time plus extension time (e.g., in a cold chamber). Insome cases, dwell time can equal ramp time plus melt time (e.g., in ahot chamber). In some cases, the melt time can be less than theextension time. Thus, dwell times based on ramp time plus extension timecan provide an upper limit for dwell times in both hot side and coldside chambers. In an example, the dwell time using the cartridge-basedthermocyclers of the present disclosure can be 5 seconds and can include3 seconds of ramp time and 2 seconds of extension time.

In various implementations, a reference to time (including, but notlimited to, cycle time, ramp time, extension time, melt time, dwelltime) in a nucleic acid amplification cycle is dependent, at least inpart, on the number of nucleic acids to be amplified, the sequence ofnucleic acids to be amplified, the oligonucleotide primers used in theamplification reaction, and any combination thereof. In one embodiment,the thermocyclers and methods provided herein are useful for amplifyinga nucleic acid comprising between about 50 base pairs (bp) and about50,000 bp, between about 50 bp and about 40,000 bp, between about 50 bpand about 30,000 bp, between about 50 bp and about 20,000 bp, betweenabout 50 bp and about 10,000 bp, or between about 50 bp and about 5,000bp. In one embodiment, the thermocyclers and methods provided herein areuseful for amplifying a nucleic acid comprising between about 50 bp andabout 5,000 bp, between about 50 bp and about 4,000 bp, between about 50bp and about 3,000 bp, between about 50 bp and about 2,000 bp, betweenabout 50 bp and about 1,000 bp, or between about 50 bp and about 500 bp.In another embodiment, the thermocyclers and methods provided herein areuseful for amplifying a nucleic acid comprising between about 50 basepairs bp and about 500 bp, between about 50 bp and about 400 bp, betweenabout 50 bp and about 300 bp, between about 50 bp and about 200 bp,between about 100 bp and about 1,000 bp, between about 100 bp and about500 bp, between about 100 bp and about 400 bp, between about 100 bp andabout 300 bp, between about 150 bp and about 1,000 bp, between about 150bp and about 500 bp, between about 150 bp and about 400 bp, or betweenabout 150 bp and about 300 bp. In one embodiment, the nucleic acidamplification for a nucleic acid template comprising from about 50 bp toabout 5,000 bp is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or more efficient. In one embodiment, a nucleic acid amplificationcycle time for a nucleic acid template comprising from about 50 bp toabout 5,000 bp is less than about 60 seconds, less than about 50seconds, less than about 40 seconds, less than about 30 seconds, lessthan about 20 seconds, less than about 10 seconds, or less than about 5seconds.

Cartridge-Based Rapid Thermocyclers

The disclosure provides systems for performing thermal cycling (also“thermocycling” herein) of a fluid (also “sample” or “sample fluid”herein). The systems herein can be used for thermocycling a sample fluidto perform biological or biochemical analysis. In some examples, thesystems herein can be used for thermocycling a sample fluid comprising adeoxyribonucleic acid (DNA) target to perform polymerase chain reaction(PCR). The systems can be cartridge-based systems (also “cartridges” or“cartridge-based thermocyclers” herein) that enable low-cost, disposablePCR systems to be realized. In some cases, the cartridges can bereal-time, multiplexed systems (also “multiplexed assays” herein). Thecartridges can be coupled to other PCR system components, such as, forexample, one or more other cartridges and/or an instrument. In someimplementations, PCR systems comprising disposable cartridge system(s)coupled with a durable instrument can be provided.

The cartridges can be configured to move fluid between distinctchambers. Each chamber can have a given temperature, composition, volumeand/or shape. Individual chambers can be heated, cooled, and/orcompressed to mix fluid within the chamber or to propel fluid in thechamber into another chamber. Further, the chambers can be shaped toinhibit trapping of air bubbles. The chambers can be configured to allowrapid thermal equilibration. The chambers have any shape that does notinterfere with the movement of fluid between chambers, which includesgenerally planar or bubble-like shapes, including hemispherical andspherical shapes.

Reference will now be made to the figures, wherein like numerals referto like parts throughout. It will be appreciated that the figures (andfeatures therein) are not necessarily drawn to scale.

FIG. 1 shows a single unit thermocycler (also “thermocycling unit”herein). The thermocycler can rapidly change the bulk temperature of asmall volume of fluid between a first temperature T₁ and a secondtemperature T₂. In some cases, T₁<T₂; for example, T₁ can be nominallyabout 55° C., about 60° C., about 65° C., or any temperature betweenabout 55° C. and about 65° C., and T₂ can be nominally about 95° C. Insome examples, T₁ can be about 55° C., 56° C., 57° C., 58° C., 59° C.,60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C.,69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C. and the like. Anydescription herein in relation to a given value of T₁ equally applies toother values of T₁ at least in some configurations. In some examples, T₂can be about 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92°C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C.,101° C., 102° C., 103° C., 104° C., 105° C. and the like. Anydescription herein in relation to a given value of T₂ equally applies toother values of T₂ at least in some configurations.

The thermocycler can comprise one or more parts. In some examples, thethermocycler can comprise a disposable portion 1 and a durable orreusable portion 2. The disposable portion (also “disposable” herein)can be provided, for example, on a single cartridge or cartridgeportion, or on (e.g., spread across) multiple cartridges or cartridgeportions. In one embodiment, the durable portion of a thermocyclercomprises a receptacle or attachment means for receiving the disposableportion. In one example, the components of the disposable portion shown1 are provided on a single cartridge. In one embodiment, this cartridgecouples to another cartridge. In a further embodiment, this cartridgecouples with an instrument or analyzer. In some cases, the disposable isused once and disposed of. For example, all parts of the disposable arediscarded. The durable or reusable portion can be provided, for example,on a durable instrument or analyzer. In some cases, the durableinstrument and at least a subset or all parts associated with can bereused through the life of the instrument. In one embodiment, forsimultaneous thermocycling of a plurality of samples, a plurality ofcartridges are used simultaneously with one reusable portion orinstrument. For example, the multiple cartridges are aligned inparallel.

In an example, the disposable comprises of a polydimethylsiloxane (PDMS)top block 3 which has cavities molded within it. Two shallow chambers,left chamber 4 (e.g., at the first temperature T₁) and right chamber 5(e.g., at the second temperature T₂), can be provided. Each chamber canhave nominal dimensions of about 12 mm in diameter and about 0.5 mm inheight. The chambers can be connected by a connecting channel 6 with alength of about 5 mm and a cross-section with a height of about 0.30 mmand a width of about 0.5 mm. The areas above the chambers can becompliant and can be deformed such that the internal volume of thechamber can be changed to as little as, for example, 10% of its originalundeformed volume of π×(12 mm)²/4×(0.5 mm) or about 56 microliters (μL).

In some examples, cartridges of the disclosure can comprise chambersthat are configured to be deformed on one side (e.g., along a topsurface of each chamber) or on two sides (e.g., along a top and a bottomsurface of each chamber). In some cases, individual chambers can bedeformed using different configurations. In some examples, thedeformation can result in a change of internal volume of the chamber toless than about 90% of its original undeformed volume, less than about80% of its original undeformed volume, less than about 70% of itsoriginal undeformed volume, less than about 60% of its originalundeformed volume, less than about 50% of its original undeformedvolume, less than about 40% of its original undeformed volume, less thanabout 30% of its original undeformed volume, less than about 20% of itsoriginal undeformed volume, less than about 10% of its originalundeformed volume, and the like.

In various implementations, the boundaries of the chambers and/or thechannels connecting said chambers are defined by any sealing means orbarrier which closes a chamber to prevent movement of fluid into or outof the chamber. In one embodiment, any chamber or component of athermocycler comprises one or more openings with sealing means to allowfor the addition or removal of gases, solids and/or fluids, e.g., aninlet or port. In one example, an opening comprises a seal or valve. Inone embodiment, a chamber and/or channel is opened by an external forceapplied to or next to the chamber and/or channel. In one example, theseal is a burstable seal. Methods to open a seal include, withoutlimitation, application of pressure, mechanical actuation, heat andchemical reaction. Barriers include those which are fixed, movable oralterable components inserted into channels of the cartridge. Barriersand seals are alternatively a component of a durable portion of athermocycling unit. In one embodiment, barriers are an external forceprovided by the durable portion of a thermocycling unit, for example, aclamp. In one embodiment, some of the channels remain open during athermocycling reaction, while others may remain closed. In one example,a channel and/or chamber is opened or closed at any time point prior toor during a thermocycling reaction. Once a barrier or seal is opened, asubstance, such as a fluid, in many implementations, is moved from onechamber to another, for example, by pressure from an actuator.

In some implementations, channels 7 and 8 can allow filling andextracting of fluid (e.g., sample) from the chambers 4 and 5,respectively, via a filling or collecting device (e.g., a syringe, orancillary chambers on the same disposable). The channels can have across-section with a height of about 0.3 mm and a width of about 0.5 mm.A plate 9 (e.g., a thin plate of glass or some other suitable material)can be bonded to the PDMS top block (e.g., using a plasma cleaningprocess). The bonding can allow very high adhesion between the twomaterials to support large internal pressures caused by vapor pressureand compression of the chamber volumes. The plate 9 can have a thicknessof, for example, about 0.14 mm.

The durable instrument 2 can interact with the disposable 1. The partsor components of the durable instrument 2 that interact with thedisposable 1 can include, for example, actuator heads 10 and 11. Theactuator heads can deform the fluidic chambers to move fluid from onechamber (e.g., the chamber held at T₁) to the other chamber (e.g., thechamber held at T₂), or vice versa, over multiple cycles (e.g., between20 and 30).

In some implementations, the plate 9 can be supported and in contactwith heater blocks 12 and 13. The heater blocks can be formed of a heatconductive material such as, for example, aluminum, copper or othermetals. The heater blocks can be kept at temperatures T₁ and T₂ byheaters 14 and 15, respectively. In some cases, the heaters 14 and 15can be thin film resistive heaters with leads 16 and 17, respectively,for providing current to each heater. In other cases, the heater blockscan be heated by other heaters 14, 15, such as, for example,thermoelectric heaters, thin film heaters, etc. The two heater blockscan be separated by an air gap to minimize temperature coupling betweenthe two chambers. Temperature probes 18 and 19 (e.g., thermocouples) canbe used to monitor the heater block temperatures. In an additionalembodiment, a means for measuring temperature, e.g., temperature probe,is coupled to or in contact with one or more regions of the disposable,for example, one or more chambers. The temperature probes can be used ina temperature control feedback loop to keep the temperatures constant attheir respective set-points (e.g., T₁ and T₂). In one embodiment, hecontrol feedback loop is provided on the durable instrument. Forexample, the thermocouple signals can be acquired by a data acquisitionboard and further processed on a processing or computing unit of thedurable instrument. Based on the temperature reading received and/orother control parameters (e.g., temperature programming, opticaldetection signal of reaction progress etc.), the durable instrumentprovide control signals to one or more components (e.g., heater voltageor current controls, actuators, etc.) in a feedback mechanism.

In yet other cases, heater blocks are not be used; instead, heating canbe provided directly to the chambers (or to a structure surrounding thechambers, such as, for example, the plate 9 and/or the top block 3, or alaminate layer on a laminated cartridge described elsewhere herein). Forexample, convective heating (or cooling) using phase change or a fluidsuch as oil, air or water can be used instead. Any description herein ofheating of chambers equally applies to cooling of chambers at least insome configurations.

In one embodiment, one or more heaters are warmed up (or alternativelycooled down) to a desired reaction temperature prior to performing athermocycling reaction. In another embodiment, one or more heaters arewarmed up (or alternatively cooled down) during the course of athermocycling reaction. In one example, a heater is warmed up to adesired temperature in less than about 10 minutes, less than about 9minutes, less than about 8 minutes, less than about 7 minutes, less thanabout 6 minutes, less than about 5 minutes, less than about 4 minutes,less than about 3 minutes, less than about 2 minutes, less than about 1minute, or less than about 30 seconds.

In some embodiments, the durable portion of a thermocycler providedherein comprises a plurality of heaters, wherein each heater providestemperature control for one or more chambers of a cartridge. Forexample, the thermocycler comprises 1, 2, 3, 4 or more heaters. Inanother embodiment, the thermocycler comprises one or more coolingelements. In one embodiment, a thermocycler comprises two heaters whichprovide temperature control for one chamber of a cartridge, for example,the cartridge is disposed between the two heaters. In another example,one or more heaters provide temperature control to one or morecartridges simultaneously, wherein the cartridges are aligned inparallel.

FIG. 2A shows a simulated profile of fluid temperature in a hot chamber(e.g., right chamber 5) immediately after fluid transfer from a coldchamber (e.g., left chamber 4). The fluid in the cold chamber isinitially held at a temperature T₁ and is moved into the hot chamber attime t=0. The fluid immediately increases in temperature and at about 5seconds the average temperature is at T₂±3° C. In this example, T₁ isapproximately 55° C. (328 K) and T₂ is approximately 95° C. (368 K).

FIG. 2B shows a histogram of temperatures of the fluid entering the hotchamber in FIG. 2A with a progression from an average of 55° C. (328 K)to an average of 95° C. (368 K) in about 5 seconds. In this example,most of the fluid volume reached a temperature within a band of 95° C.(368 K)±3° C. within only about 5 seconds.

In various embodiments, the cartridges of the disclosure allow veryshort dwell times for each thermal cycle to be achieved. This can be animportant factor in establishing a fast time-to-result PCR test. Thetimescales of molecular biological reactions associated with PCR can bemuch shorter than 1 second. Therefore, the time-determining factor inrapid thermocycling can be the length of dwell time for each thermalcycle. In this example, for 20 cycles at about 5 second dwell times ineach chamber (cycle time of about 10 seconds), the total thermocyclingtime can be about 200 seconds (3.3 minutes). Most commercialthermocyclers (e.g., ABI 7900) require 30 minutes to an hour to run thisPCR because of the length of time needed to heat and cool the thermalmass of these systems (e.g., disposable tubes and/or plates held inmetal blocks). Thus, PCR with a given PCR efficiency (e.g., 92% PCRefficiency) can be completed within a total thermocycling time that isat least 9 times (e.g., 30×60 seconds/200 seconds=9) shorter than thecorresponding thermocycling time on a conventional system. In anotherexample, a Roche LightCycler II 480 Real Time PCR System can requireabout 23 minutes to complete 20 cycles, while a cartridge with two-sidedheating and dwell times of about 3 seconds, described in greater detailelsewhere herein, can perform this PCR in about 2 minutes (e.g., 20×3seconds×2=120 seconds=2 minutes), or about 11.5 faster (e.g., 23minutes/2 minutes=11.5). In some examples, cartridges of the disclosurecan complete a PCR with a given PCR efficiency within a totalthermocycling time that is shorter than the corresponding (e.g., havingthe same PCR efficiency) thermocycling time on a conventional system bya factor of at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 9.5, at least about 10,at least about 10.5, at least about 11, at least about 11.5, at leastabout 12, at least about 12.5, at least about 13, at least about 14, atleast about 15, or more. In some examples, the PCR efficiency can be atleast about 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, and the like.In an example, PCR with a PCR efficiency of at least 92% is completedwithin a total thermocycling time that is at least about 9 times shorterthan the thermocycling time on a conventional system with the same PCRefficiency. In another example, PCR with a PCR efficiency of at least92% is completed within a total thermocycling time that is at leastabout 11.5 times shorter than the thermocycling time on a conventionalsystem with the same PCR efficiency.

In some examples, the total thermocycling time can be less than about 10minutes, less than about 8 minutes, less than about 6 minutes, less thanabout 5 minutes, less than about 4 minutes, less than about 3 minutes,less than about 2 minutes, less than about 1 minute, less than about 0.5minute, and the like. In some examples, a cartridge-based thermocyclerhas a total thermocycling time of about 5 minutes, about 4 minutes,about 3 minutes, about 2 minutes, about 1 minute, about 0.5 minute, orless. In other examples, the total thermocycling time to achieve a PCRefficiency of at least 85% is less than about 15 minutes, less thanabout 10 minutes, less than about 9 minutes, less than about 8 minutes,less than about 6 minutes, less than about 5 minutes, less than about 4minutes, less than about 3 minutes, less than about 2 minutes, less thanabout 1 minute, less than about 0.5 minute, and the like. In otherexamples, the total thermocycling time to achieve a PCR efficiency of atleast 90% is less than about 15 minutes, less than about 10 minutes,less than about 9 minutes, less than about 8 minutes, less than about 6minutes, less than about 5 minutes, less than about 4 minutes, less thanabout 3 minutes, less than about 2 minutes, less than about 1 minute,less than about 0.5 minute, and the like. In other examples, the totalthermocycling time to achieve a PCR efficiency of at least 91% is lessthan about 15 minutes, less than about 10 minutes, less than about 9minutes, less than about 8 minutes, less than about 6 minutes, less thanabout 5 minutes, less than about 4 minutes, less than about 3 minutes,less than about 2 minutes, less than about 1 minute, less than about 0.5minute, and the like. In other examples, the total thermocycling time toachieve a PCR efficiency of at least 92% is less than about 15 minutes,less than about 10 minutes, less than about 9 minutes, less than about 8minutes, less than about 6 minutes, less than about 5 minutes, less thanabout 4 minutes, less than about 3 minutes, less than about 2 minutes,less than about 1 minute, less than about 0.5 minute, and the like. Inother examples, the total thermocycling time to achieve a PCR efficiencyof at least 95% is less than about 15 minutes, less than about 10minutes, less than about 9 minutes, less than about 8 minutes, less thanabout 6 minutes, less than about 5 minutes, less than about 4 minutes,less than about 3 minutes, less than about 2 minutes, less than about 1minute, less than about 0.5 minute, and the like.

The cartridges of the present disclosure can be used as multiplexedassays. In one aspect, a thermocycling unit provided herein comprises oris operably connected to a detector. For example, one or more componentsof a cartridge are configured to enable detection of a sample within thecomponent. For example, the detector detects the existence of an analytein the sample or the amount of a signal indicative of a characteristicof the sample. Signals include, without limitation, luminescence,fluorescence, turbidity, radioactivity and electrical currents. In anexemplary embodiment, a nucleic acid analyte is detected using adetectable label. Exemplary labels include, without limitation,radiolabels, intercalating dyes, enzymes, haptens, chemiluminescentmolecules, and fluorescent molecules. In some implementations, one ormore regions of the cartridge (e.g., one or more of the chambers, aregion in the fluid flow path between chambers, etc.) can be monitoredto detect amplification of the target DNA (e.g., using optical or otherdetection methods, such as bioimpedance and colorimetry). In some cases,the detection can be implemented through optical multiplexing by usingone or more fluorescent probes. In one example, each target DNA sequencecan be detected by a fluorescent label (also “fluorophore” herein), witha different label corresponding to each target. In another example,multiple labels can be applied to each target DNA sequence. Thedetection can be performed in real-time. For example, multiplexedreal-time PCR can be used to identify the presence and/or the quantityof particular sequences of DNA.

Further, the thermocycling unit can be reproduced multiple times on amore complicated cartridge (e.g., a disposable cartridge) or cassette.For example, multiple thermocycling units can be deployed within acartridge to perform a multiplexed assay. In some cases, at least asubset or all of the thermocycling units can be identical. In othercases, one or more of the thermocycling units can be unique (e.g., eachthermocycling unit can have a different configuration including, but notlimited to, chamber shape, volume, temperature etc.). In someimplementations, one or more of the thermocycling units can have adedicated detector. For example, each of the thermocycling units canhave a dedicated detector. Alternatively, at least a subset of thethermocycling units can share a detector. For example, eachthermocycling unit can have a switching element in front of a timemultiplexed detector. Individual detectors can be suitable or configuredfor detecting PCR on one or more of the thermocycling units.

The cartridges of the present disclosure can comprise additionalcartridge portions or be coupled to one or more other cartridges. Forexample, individual thermocycling units can be linked to one or morereaction chambers (e.g., on the additional cartridge portion or onanother cartridge) for implementing sample preparation. In some cases,multiple thermocycling units can be linked to a single set of reactionchambers for implementing sample preparation. In other cases, multiplethermocycling units can be linked to multiple sets of reaction chambersfor implementing sample preparation. In an example, a first subset ofthermocycling units can be linked or connected to a first set ofreaction chambers while a second subset of thermocycling units can belinked or connected to a second set of reaction chambers. In anotherexample, one or more individual thermocycling units can each be linkedto its own set of reaction chambers. Each set of reaction chambers caninclude, for example, 1, 2, 3, 4, 6, 8, 10 or more reaction chambers.

Starting volume (also “sample starting volume” or “sample volume”herein) can be important in PCR (e.g., for high sensitivity PCRreactions). At low target analyte concentration, a larger sample volumecan facilitate detection by increasing probability of the analyte beingpresent in the sample for PCR analysis. Because of sample compositionvariability (e.g., in real human samples such as urine or saliva), thesample can be processed through a pre-filter to remove, for example,solid matter (e.g., insoluble material) prior to a purification step. Inone aspect, the cartridges provided herein comprise or are coupled to achamber or vessel for sample preparation. In one example, the sample isprocessed prior to addition to a cartridge or processed, in whole or inpart, in one or more chambers or components of a cartridge. A first stepin purification can be to lyse all the organisms of interest (e.g.,bacteria, viruses, etc.) to release total nucleic acid. A next step inpurification can involve a solid phase material (e.g., filter or beads)with an affinity for the nucleic acid or molecule of interest. Afteraffinity capture of the nucleic acid to the solid phase material, thenucleic acid can be washed with a wash solution prior to elution withwater. The elution can contain the purified nucleic acid to be used forPCR analysis. Using as much of the elution as possible for PCR can bedesirable in order to increase or maximize the sensitivity. For example,a PCR starting volume of more than about 25 μL (e.g., 50 μL) can be usedto achieve improved sensitivity. This can allow the PCR to proceed withlow target concentrations (e.g., 10 copies/μL). Therefore, rapidthermocyclers of the disclosure can be configured to provide anincreased heat transfer rate (° C./second) while ensuring that this heattransfer rate can be achieved with a reasonable starting volume. In oneembodiment, a sample preparation chamber or vessel comprises, or isconnected to an auxiliary chamber which comprises, sample preparationreagents such as Lysozyme or Proteinase K. In one embodiment, a samplepreparation chamber or vessel comprises, or is connected to an auxiliarychamber which comprises, a solid support for immobilizing an analyte,e.g., nucleic acid, in the sample. Solid supports include magneticsupports, such as beads, that can be manipulated by a magnetic field. Inanother embodiment, a sample preparation chamber or vessel is connected,directly or indirectly, to a waste chamber for collecting samplematerial which interferes with an amplification reaction (e.g., cellpellet).

A cartridge-based thermocycler of the present disclosure can have anysuitable starting volume, such as at least about 25 μL, at least about30 μL, at least about 35 μL, at least about 40 μL, at least about 45 μL,at least about 50 μL, at least about 55 μL, at least about 60 μL, atleast about 65 μL, at least about 70 μL, at least about 75 μL, at leastabout 80 μ, at least about 85 μL, at least about 90 μL, at least about95 μL, at least about 100 μL, and the like. In some examples, acartridge-based thermocycler has a starting volume of about 25 μL, 30μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80μL, 85 μL, 90 μL, 95 μL, 100 μL or more. In one embodiment, one or morechambers of a cartridge has a non-compressed volume capacity of at leastabout 10 μL, at least about 15 μL, at least about 25 μL, at least about30 μL, at least about 35 μL, at least about 40 μL, at least about 45 μL,at least about 50 μL, at least about 55 μL, at least about 60 μL, atleast about 65 μL, at least about 70 μL, at least about 75 μL, at leastabout 80 μL, at least about 85 μL, at least about 90 μL, at least about95 μL, at least about 100 μL, and the like. In another embodiment, oneor more chambers of a cartridge has a non-compressed volume capacity ofat least about 50 μL, at least about 100 μL, at least about 150 μL, atleast about 200 μL, at least about 250 μL, at least about 500 μL, andthe like. In one embodiment, one or more chambers of a cartridge has acompressed volume of less than about 100 μL, less than about 50 μL, lessthan about 40 μL, less than about 30 μL, less than about 20 μL, lessthan about 10 μL, or less than about 5 μL. The connecting channeldisposed between two chambers has a volume, for example, of about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 20% of the total fluid volume ofthe two chambers. For example, from about 1 μL to about 50 μL.

A cartridge-based thermocycler of the present disclosure can have aproduct rate of at least about 10 μL° C./second, at least about 25 μL°C./second, at least about 50 μL° C./second, at least about 75 μL°C./second, at least about 100 μL° C./second, at least about 150 μL°C./second, at least about 200 μL° C./second, at least about 250 μL°C./second, at least about 300 μL° C./second, at least about 325 μL°C./second, at least about 350 μL° C./second, at least about 375 μL°C./second, at least about 400 μL° C./second, at least about 425 μL°C./second, at least about 450 μL° C./second, at least about 500 μL°C./second, at least about 550 μL° C./second, at least about 600 μL°C./second, at least about 650 μL° C./second, at least about 700 μL°C./second, and the like. In some examples, a cartridge-basedthermocycler has a product rate of about 10 μL° C./second, about 25 μL°C./second, about 50 μL° C./second, about 75 μL° C./second, about 100 μL°C./second, about 150 μL° C./second, about 200 μL° C./second, about 250μL° C./second, about 300 μL° C./second, about 325 μL° C./second, about350 μL° C./second, about 375 μL° C./second, about 400 μL° C./second,about 425 μL° C./second, about 450 μL° C./second, about 500 μL°C./second, about 550 μL° C./second, about 600 μL° C./second, about 650μL° C./second, about 700 μL° C./second, or more. In one example, astarting volume of 50 μL with a heating or cooling rate of about 35° C.in 5 seconds is used. In this example, a product rate of about 350 μL°C./second is achieved. In some examples, a cartridge-based thermocyclercan have a product rate within a range of about 10-700 μL° C./second,about 25-700 μL° C./second, about 100-700 μL° C./second, about 10-450μL° C./second, about 25-450 μL° C./second, about 100-450 μL° C./second,about 300-400 μL° C./second, and the like. In some examples, acartridge-based thermocycler can have a product rate within a sub-range.For example, the sub-range can be about (or at least about): 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of a range (e.g., arange of about 10-700 μL° C./second, about 25-700 μL° C./second, about100-700 μL° C./second, about 10-450 μL° C./second, about 25-450 μL°C./second, about 100-450 μL° C./second, about 300-400 μL° C./second, andthe like). In some cases, the sub-range can comprise a lower portion ofa range, an upper portion of a range, or an interior portion of a range.In some cases, the sub-range can have a width of at least about 0.01 μL°C./second, at least about 0.1 μL° C./second, at least about 1 μL°C./second, at least about 2 μL° C./second, at least about 5 μL°C./second, at least about 10 μL° C./second, at least about 20 μL°C./second, at least about 50 μL° C./second, at least about 100 μL°C./second, at least about 150 μL° C./second, at least about 200 μL°C./second, at least about 250 μL° C./second, and the like.

A cartridge-based thermocycler having a suitable starting volume canhave a cycle time of less than about 20 seconds, less than about 15seconds, less than about 12 seconds, less than about 11 seconds, lessthan about 10 seconds, less than about 9 seconds, less than about 8seconds, less than about 7 seconds, less than about 6 seconds, less thanabout 5 seconds, less than about 4 seconds, and the like. In someexamples, a cartridge-based thermocycler has a cycle time of about 12seconds, about 11 seconds, about 10 seconds, about 9 seconds, about 8seconds, about 7 seconds, about 6 seconds, about 5 seconds, about 4seconds, or less.

The PCR chambers 4 and 5 can be coupled to one or more other chambers insome implementations. In an example, the disposable cartridge can beformed with three chambers. An additional or auxiliary chamber (notshown) can have nominal dimensions of about 12 mm in diameter and about0.5 mm in height. In some examples, the auxiliary chamber can be apre-chamber connected, permanently or temporarily (e.g., before beingsealed off), to one or more PCR chambers of the disclosure. Theauxiliary chamber can be capped by a thin flexible membrane material.The auxiliary chamber (also “blistered” chamber herein) can containlyophilized reagents of Lysozyme and Proteinase-K stored in dry (e.g.,powder) form. The auxiliary chamber may or may not be heated (e.g., by aheater on the durable instrument). In one example, a heater on a surfaceof the auxiliary chamber (e.g., on a back side of the auxiliary chamber)can apply heating needed for heat activation in the auxiliary chamber.The membrane can be blistered into the volume of the auxiliary chamberto move fluid to the next series of chambers representing the PCRthermocycling stage (e.g., chambers 4 and 5). The blistering can bedone, for example, by an external actuator (e.g., an actuator on thedurable instrument). In some cases, the actuator can execute minutemovements (e.g., movements of about 0.1 mm) to mix the contents of theauxiliary chamber. The auxiliary chamber can be compressed by anactuator to move fluids to downstream processes. For example, thecontents of the auxiliary chamber can be pushed out by the actuatordeforming the blisterable membrane into the hot chamber (e.g., chamber5) in the thermocycling stage. In some examples, the cartridge cancomprise a valve that seals the contents (e.g., the sample fluid) in thehot thermocycling chamber. In one embodiment, the blistered chambercomprises reagents useful for performing a nucleic acid-basedamplification reaction and/or detection of nucleic acid amplificationproducts. Exemplary reagents include, without limitation, ahybridization oligonucleotide (e.g., probe, primers), ions and buffers.In one example, the blistered chamber comprises reagents for samplepreparation. In another example, the blistered chamber compriseslyophilized or otherwise dried reagents.

In some examples, the contents can be sealed in the thermocyclingchambers (e.g., chambers 4 and 5) via one or more pinch or clamp points,described in greater detail elsewhere herein. The pinch points can bepinched by, for example, one or more components (e.g., actuators orpistons) on the durable instrument. The pinch points can be clamped. Insome cases, one or more valves are used (e.g., to control fluid and/orgas flow, such as to close and/or open channels to chambers). In somecases, the valve can be used instead of the pinch point. In some cases,the valve can be used in combination with the pinch point. In somecases, one or more valves can be combined with one or more pinch points.

The hot (thermocycling) chamber can be heated (e.g., maintained at about95° C.) by an external thin film heater. Upon receiving the sample fluidfrom the auxiliary chamber, the contents of the hot chamber can beelevated in temperature for a given period of time (e.g., 95° C. forabout 1 minute). This can inactivate the Proteinase-K and further lysethe cell walls of bacteria not lysed by the Lysosyme, thereby releasingDNA sample in the sample fluid from cell nuclei and disabling inhibitoryfactors to PCR. An actuator can be used to compress the hot chamber tomove or push the sample fluid (e.g., contents of chamber 5) to the coldchamber (e.g., chamber 4) in the thermocycling stage.

The cold (thermocycling) chamber may or may not be heated (e.g.,maintained at about 65° C.). Lyophilized “Master Mix” reagents includingprimers and TaqMan® probes can be stored in the cold chamber (e.g.,maintained at about 65° C.). After mixing with the Master Mix, thesample fluid can be ready for thermocycling. An actuator can be used tocompress the cold chamber to move or push the sample fluid back to thehot chamber to begin the thermocycling processes of the disclosure.

Thus, cartridge-based rapid thermocyclers having one or more auxiliarychambers can be provided. The auxiliary chambers can be used, forexample, for filling, emptying or regulation of sample fluid in the PCRchambers. In an example, an auxiliary chamber can be used for pre-PCRpreparation of the sample.

Laminated Disposable Cartridge

In some implementations, the cartridge comprising the chambers can havea laminate construction. The laminated cartridge can be disposable. Forexample, the laminated cartridge can be provided as a disposable elementof a low-cost PCR system. In some cases, the laminated cartridge can beused strictly for PCR without addressing pre-PCR preparation of thesample. Alternatively, at least a portion of pre-PCR preparation can beprovided on the laminated cartridge. Cartridges of the disclosure,including laminated cartridges, can be used in concert with mechanicalactuation for moving a fluid from one chamber to another. The fluid ineach chamber can be rapidly brought to desired temperatures for PCR(e.g., within 5 seconds). In some examples, each chamber can be held ata given PCR temperature (e.g., 95° C. or 65° C.) using fixed-temperatureheater blocks. Other examples of heating configurations are described ingreater detail elsewhere herein. Further, the cartridges enable thefluid sample to be optically interrogated for a fluorescence signal.Low-cost, disposable materials can be used to construct robustcartridges that can resist high pressures of PCR. Any aspects of thedisclosure described in relation to laminated cartridges equally appliesto other cartridges of the disclosure at least in some configurations.

FIG. 3 provides an example of a laminated cartridge C comprising layeredsheets or membranes 31 and 32 (e.g., formed from plastic or anothersuitable material) and pressure sensitive adhesive (PSA), collectivelyreferred to as laminate layers herein. The PSA can be provided as layers33 and 34 (also “PSAs” herein) between the layered sheets of plastic 31and 33. The PSA can be used to bond two outer membranes (e.g., plasticmembranes) together. Other examples of techniques that can be used tobond laminate layers include use of other adhesives (e.g., liquid orsolid adhesives), fusing the laminate layers together through (e.g.,heat, ultrasound), etc.

The PSA layers can each have a given thickness. The thickness of thePSAs (e.g., combined thickness of the PSA layers) can define internalvolumes of the PCR chambers 4 and 5. For example, the membranes 31 and32 can form upper and lower surfaces, respectively, of each chamber, andthe PSAs can form side walls of each chamber having a heightcorresponding to the combined thickness of the PSAs. In some cases,additional layered sheets (e.g., plastic layers) can be provided incombination with the PSAs. For example, additional plastic layers can beadded to increase the fluid holding volumes of the chambers. In somecases, the volumes of the chambers can be identical. In other cases, thevolumes of the chambers can differ.

The laminated cartridge can be heated on one or more surfaces. Forexample, at least one of the membranes 31, 32 (e.g., bottom membrane 32)can be positioned adjacent to heaters or adjacent to heater blocks (notshown) and formed of a thermally conductive material (e.g., polyimide,or polyester) configured for efficient heat transfer such that fluidthat enters each chamber is rapidly equilibrated at a desiredtemperature.

The device geometry can be configured for efficient heat transfer andcost by changing thicknesses of the layered sheets or membranes 31 and32 (e.g., thicknesses of the plastic). For example, a thinner plasticlayer which is less durable provides improved heat transfer rates to thechambers. Further, the device geometry can be configured for efficientheat transfer and cost by changing heights and diameters of the PCRchambers. As described above, heights of the PCR chambers can bechanged, for example, by changing the number and thickness of individuallaminate layers that make up the sides of each chamber. The diameters ofthe PCR chambers can be changed by, for example, providing PSA layerswith cutouts of different diameters. In one example, as shown in FIG. 3,the cutouts can be substantially circular. In another example, as shownin FIGS. 4, 5 and 6, the cutouts can be substantially oval. In somecases, the cutouts and resulting chambers can be shaped (e.g., in a teardrop shape) in order to decrease trapping of air or vapor bubbles in thefluid being cycled on the cartridge. In some cases, chambers and/orfluid flow paths can be shaped (e.g., in a tear drop shape) to avoid orreduce re-entrant or sharp corners and/or steps that can serve asnucleation sites during filling and/or cycling of fluid on thecartridge. In an example, the chambers and/or fluid flow paths can beconfigured such that their surface maintains a curvature that is largerthan a curvature of the wavefront of the fluid flowing across thesurface. Further, the cutouts can include various features, such as theconnecting channel 6 and the channels 7 and 8 for filling/extractingfluid (e.g., sample) to/from the chambers 4 and 5. In some cases, thechannels 7 and 8 can be used for filling, extracting and/or venting. Forexample, channel 7 is useful as a filling channel for filling (e.g., offluid) and channel 8 is useful as a venting channel for venting (e.g.,to prevent gases from being trapped in the device while filling), andvice versa. In one embodiment, the channel that is used for ventingconnects the respective chamber 5 to outside air. For example, thechannel 8 can connect the chamber 5 to the ambient environment. Asdescribed elsewhere herein, fill or vent ports (e.g., ports 45 in FIG.4), can be used for connecting to the channels 7 and/or 8. In somecases, a fill port can be used for extraction of fluid. Fill/extractionports and vent ports may or may not have the same configuration. In anexample, the port exposed to the surroundings (e.g., outside air) has agas permeable seal (not shown) to allow air in but to keep the devicefrom being contaminated. In another example, the port exposed to thesurroundings can comprise a check valve for letting gas out from thechamber (e.g., via the channel) while not allowing air or othercontaminants into the chamber. After filling and/or venting, eachchannel can be closed to encapsulate the fluid during thermocycling.

In some implementations, one or more of the materials comprising thelaminate layers of the cartridge can have hydrophilic properties. Forexample, plastic materials (e.g., plastic used to form one or more ofthe membranes) can have hydrophilic properties. The hydrophilic natureof the materials can provide various advantages for cartridge operation.For example, cartridge chambers (and/or fluid flow paths) havinghydrophilic surfaces can be filled without leaving behind small trappedair or vapor bubbles. Such bubbles can become nucleation sites forlarger air or vapor bubbles as the cartridge is heated for PCR. In somecases, this can lead to lower PCR efficiency. In some cases, the amountof air or vapor bubbles trapped during filling can be decreased by usingcartridges with chambers (and/or fluid flow paths) having hydrophilicproperties. Further, bubble formation can cause signal dropouts duringin the detection system (e.g., during optical detection). Hydrophilicproperties can be used in concert with the previously described shapedchambers and/or shaped fluid flow paths to achieve desired fluid flowconditions during filling and cycling on the cartridge.

FIG. 13 shows fluid contact angles of a 10 μL water droplet on variousmaterial surfaces. Examples of cartridge materials with differenthydrophilic properties include plastic materials such as untreated PDMS,treated PDMS, Kapton® MT, raw polyethylene terephthalate (PET), coatedPET, glass, etc. Some materials (e.g., plastic materials) are furthermodified with coatings (e.g., Vistex 111-50) and/or by chemical orphysicochemical treatment (e.g., plasma treatment) to make them morehydrophilic. In some implementations, a given amount (e.g., a smallamount that does not undesirably alter optical or kinetic properties ofthe sample fluid) of a detergent or other chemical material can be addedto the sample fluid (e.g., used in an assay buffer). In some cases, thechemical material (e.g., Tween-20, Bovine Serum Albumin, polyethyleneglycol) can alter sample fluid properties to enable easier filling ofthe cartridge (e.g., with less trapped bubbles, less spill, etc.). Insome examples, cartridge materials can be chosen to provide one or morefluid surfaces with a given contact angle. In one example, PET can beused instead of PDMS due to its more hydrophilic properties. In anotherexample, one or more materials with a contact angle of less than about5°, less than about 10°, less than about 15°, less than about 20°, lessthan about 30°, less than about 40°, or the like can be used. In someexamples, some surfaces or materials of the cartridge (e.g., materialsor surfaces wetted by the sample fluid) can comprise a material with asmaller contact angle (i.e., more hydrophilic) than other materials onthe cartridge.

Further, the laminated cartridge can comprise portions configured forefficient stretching upon mechanical actuation. For example, at leastone of the membranes 31, 32 (e.g., top membrane 31) can be configured tostretch efficiently to allow the fluid to be moved from one chamber toanother using mechanical actuation. In some cases, the membrane canstretch or “blister”.

The cartridge and chambers can be held against the heaters or the heaterblocks by the pressures imposed by the actuators. For example, in aconfiguration where heating is provided by heater blocks adjacent thethermally conductive bottom 32, the cartridge can be held down againstthe heater blocks (e.g., heater blocks 12 and 13). Significantcounter-pressure can be exerted by the actuator adjacent to the chamberfilled with PCR fluid (i.e., the actuator located on the same side orportion of the cartridge as the chamber that is filled with PCR fluid).

The cartridge can be provided as a rigid structure. Alignment features38 can be used to mechanically hold the individual laminate layers inplace with respect to each other. The alignment features can include,but are not limited to, screws, nuts and bolts, heat stakes, pegs,adhesive filling, etc. In some implementations, an outer casing can beprovided to allow easier handling and alignment (e.g., when insertingthe cartridge into a durable instrument or analyzer). The outer casingcan also provide features for filling the device and for valving thefluid to prevent leaking during PCR (e.g., using fill/vent ports andpinch points, as described elsewhere herein). The outer casing can becoupled to the laminate structure using, for example, the alignmentfeatures 38. In some cases, the outer casing can be coupled to thelaminate structure without using the alignment features 38. For example,the outer casing can have flanges for gripping the laminate structure.The outer casing and the laminate structure can also be provided withmating features (clips, clasps, connectors, heat stakes, snap locks,etc.) for forming a secure mechanical connection. In some cases, theouter casing can comprise one or more secondary rigid structures orcovers. For example, a first rigid cover 35 can be provided. In someexamples, a second rigid cover 36 (e.g., as shown in FIG. 3) can beprovided. In some configurations, the rigid cover can be configured toprovide adequate heat transfer. In some cases, cartridges can compriseone or more alignment fixtures (e.g., alignment fixture 48 in FIG. 4).The alignment fixture may or may not be a part of the outer casing. Thealignment fixture can be used for assembly and/or manufacturing. In someimplementations, the alignment fixture can serve as a protective coverwithout providing rigid structure. In some implementations, thealignment fixture can be used for assembly/manufacturing and does notserve as a protective cover. For example, rigid covers and/or protectivecovers are not useful on surfaces where the cartridge directly contactsheater block(s). In further implementations, the alignment fixture canprovide rigid structure. In yet other implementations, the alignmentfixture can have an optical function, as described in greater detail inrelation to FIG. 15.

In some examples, a cardboard sheet (or a sheet or layer of any othersuitable material, including disposable and/or biodegradable polymers,paper and pulp) can be coupled (e.g., bonded to, snapped onto, clippedonto, etc.) to the laminated structure for support. In some cases, thecardboard sheet can be formed as front and/or back covers. In othercases, the cardboard sheet can be shaped differently from the laminatedcartridge. For example, the cardboard sheet can be rectangular andsupport a laminated structure with a more complex shape (e.g., as shownin FIG. 4). Further, the cardboard sheet(s) can be tightly or looselycoupled to the laminated cartridge. The cardboard sheet(s) can cover anarea that is larger, equal to or smaller than the projected area of thelaminated cartridge. For example, the cardboard sheet(s) can providesupport to only a portion of the laminated cartridge. In furtherexamples, a cardboard box can be used to protect the laminated structure(e.g., as outer casing). Implementations of the laminated cartridge(e.g., with or without outer casing, cardboard backing, etc.) may be alldisposable. In some implementations, the laminated structure can beseparable from its support structure (e.g., backing and/or casingcomponents). For example, the laminate structure can be all disposablewhile at least a portion of the support structure can be reusable. Insome cases, the support structure can serve to couple the laminatedcartridge to one or more other cartridges and/or to a durableinstrument.

The laminated cartridge can comprise portions configured fortransmitting optical signals. For example, at least one of the membranes31, 32 (e.g., top membrane 31) can be positioned adjacent to opticalexcitation device(s) and detector(s) and formed of an opticallytransparent or clear material configured for transmitting opticalsignals 40 incoming to and outgoing from the sample. For example, anoptically clear top membrane 31 can be used to transmit light from alight source to the sample (e.g., optical excitation) and to transmitlight from the sample to a detector or readout (e.g., fluorescenceemission).

In some implementations, the sample fluid can be optically detected inone or more of the PCR chambers (e.g., chambers 4 and 5). For example,fluorescence excitation and readout of the fluid can be accomplished byproviding an optically transparent layered sheet or membrane formed of amaterial that allows excitation and readout to be made directly in a PCRwell (also “chamber” herein) through the sheet or membrane. For example,a plastic or polymeric material such as polyester or PET can be used asthe optically transparent material. In some cases, one or more laminatelayers (e.g., the layered sheet or membrane, covers, etc.) can bepartially formed from an optically transparent material (e.g., see FIG.4). For example, the optically transparent material can be provided inregions of the laminate layers that are located directly in an opticalpath to/from the well.

In other implementations, the sample fluid can be optically detectedoutside of the chamber(s), such as, for example, within any of the fluidflow paths (e.g., see FIGS. 4, 5 and 7). In one embodiment, a separatechamber forms within the fluid flow path (e.g., in the fluid channel 6between the fluid chambers 4 and 5) and a corresponding optical windowcan be created in the laminate structure to interrogate the fluid in theseparate chamber (collectively referred to as a “cuvette” herein). Inthis configuration, an optically transparent layered sheet or membraneis not needed, as the optical window can provide a direct optical pathto the separate chamber. An advantage of the optical window can be, forexample, that both the top membrane 31 and the bottom membrane 32 can beformed of thermally conductive materials, thus enabling two-sidedheating without loss of optical detection capability. In some cases,sample interrogation in the separate chamber enables optical detectionwith higher resolution (e.g., due to size of volume interrogated,turbulence intensity, etc.). The optical window can be adapted to fitvarious form factors (e.g., flat form factor).

In yet other implementations, combinations of the above configurationscan be used. For example, an optically transparent layer is useful tointerrogate the fluid in the separate chamber without the need for aseparate optical window (e.g., enabling a substantially flat formfactor).

With continued reference to FIG. 3, the cartridge can be configuredwithout a separate optical window or cuvette. In this configuration,optical readout can be made directly in one (or both) of the wells(e.g., wells 4 and 5). For example, optical excitation/readout can beprovided to/from one (or both) of the chambers 4 and 5 through theactuation buttons 37 and 39, respectively. In some examples, theactuation buttons 37 and/or 39 can be at least partially formed of anoptically transparent material. In other examples, the actuation buttons37 and/or 39 can comprise one or more conduits for transmitting opticalsignals to and from the well(s).

FIG. 4 is an exploded view of separate layers of the layered cartridge Cthat includes an embedded optical window 43 with portions 43 a, 43 b, 43c, 43 d, 43 e, 43 f, 43 g and 43 h. The optical window provides anoptical path to a chamber 44 located in a fluid path (e.g., a fluid flowdefined within the combined PSA layers 33, 34 between the membranes 31and 33). In one configuration, the chamber 44 is located in the fluidpath 6 between the fluid chambers 4 and 5. Together, the optical window43 and the chamber 44 can form a cuvette 43, 44.

In this example, a secondary rigid outer structure 35 and an alignmentfixture 48 (e.g., for assembly/manufacturing) can be bonded with PSA.The secondary rigid outer structure 35 can serve as a front cover. Inthis example, a rigid bottom cover is not provided as the bottom of thelaminate cartridge is placed directly onto heater block(s). Thealignment fixture 48 may or may not serve as a (protective) bottomcover. In some examples, the positions of the rigid cover 35 and thealignment fixture 48 can be reversed (e.g., depending on which cartridgesurface(s) are heated). In some examples, the rigid cover 35 can besubstituted by an alignment fixture or removed altogether (e.g., toenable two-sided heating). In some examples, the alignment fixture 48 isnot provided. PSA layer 41 can be used between the front cover 35 andthe front membrane 31. A PSA layer 42 can be used between the alignmentfixture 48 and the back membrane 32. In other examples, the PSA layerscan be bonded using other techniques known in the art, as describedelsewhere herein.

With continued reference to FIG. 4, the cartridge laminate layers canfurther comprise one or more fill or vent ports 45 with portions 45 a,45 b, 45 c, and 45 d provided, for example, on the laminate layers 35,41, 31 and 33, 34, respectively. Fill/vent ports can be provided for asubset of cartridge chambers, or for all cartridge chambers (e.g., bothof the chambers 4 and 5). Each fill/vent port 45 can be used to fill acorresponding chamber with fluid, to withdraw fluid from a correspondingchamber, to vent pressurized gas and/or fluid from a correspondingchamber, or any combination thereof. The fill/vent ports can be in fluidcommunication with one or more other chambers (e.g., a pre-PCRpreparation chamber, filter chamber, waste chamber, etc.), a syringeneedle, external or internal tubing, or any combination thereof. In someexamples, more than one fill/vent port can be provided per chamber toenable multiple fluid connections with different cartridge or externalcomponents. In some implementations, access to the fill/vent port (andthus the corresponding chamber) can be enabled from top and/or bottomsurfaces of the cartridge. For example, the fill/vent port can span oneor more laminate layers (e.g., from the top cover 35 to the combined PSAlayers 33, 34). In other implementations, the fill/vent port can beprovided within a subset of the laminate layers. For example, fillingand/or venting access can be enabled from a side surface of thecartridge.

The cartridge laminate layers can comprise one or more pinch points 46with portions 46 a, 46 b and 46 c provided, for example, on the laminatelayers 35, 41 and 33,34, respectively. Pinch points can be provided fora subset of cartridge chambers, or for all cartridge chambers (e.g.,both of the chambers 4 and 5). Each pinch point 46 can be used to seal acorresponding chamber (e.g., after completion of fluid filling, fluidwithdrawal, gas or fluid venting, etc.). The sealing can be permanent orreversible. In an example, one of the chambers can be sealed afterfilling, while another one of the chambers can be periodically vented.Prior to pinching or compression (e.g., by an actuator on a durableinstrument), the pinch points on the chambers 4 and/or 5 can providefluid communication between the chambers and the fill/vent ports 45 viathe channels 7 and/or 8, respectively. The pinch points can also providefluid communication with one or more other chambers (e.g., a pre-PCRpreparation chamber, filter chamber, waste chamber, etc.) or external orinternal tubing instead of, or in addition to the fill/vent ports 45. Insome examples, more than one pinch point can be provided per chamber toenable closing of fluid connections with different cartridge or externalcomponents. In some implementations, pinching or compression of thepinch point can be enabled from top and/or bottom surfaces of thecartridge. For example, the pinch point can be compressed through one ormore laminate layers (e.g., from the top cover 35 to the combined PSAlayers 33, 34, from the alignment fixture 48 to the combined PSA layers33, 34, or both). The pinch point can be pinched or compressed bylocating the cartridge adjacent to one or more actuators. In someexamples, the actuator(s) can pinch or compress the pinch point(s) froma top surface of the cartridge, from a bottom surface of the cartridge,or both.

In configurations comprising the cuvette, the size and/or shape of theoptically interrogated volume enables adequate optical detection. In oneembodiment, the optically interrogated volume is configured to decreaseor minimize its contribution to dead volume of PCR (e.g., fluid volumethat is not brought to temperature during any one PCR cycle). In someexamples, the interrogated volume outside of one or more PCR chambers(e.g., outside of a heated PCR chamber) can be less than about 5% orless than about 10% of the total PCR fluid volume in the cartridge.

FIG. 5 shows front, back and side views of the assembled laminatedcartridge C with the optical window 43 embedded. Together with thechamber 44, the optical window can form the cuvette 43, 44. Thecartridge further comprises the chambers 4 and 5 with respectivefill/vent ports 45 and respective pinch points 46.

FIG. 15 shows the cartridge C (e.g., a polyester cassette) with opticalcomponents. The optical window 43 can be used for interrogating a samplevolume (e.g., to direct light into an interrogation volume or samplechannel, such as the chamber 44 in the sample channel 6). In some cases,the optical window 43 and the interrogated volume can form the cuvette(e.g., cuvette 43, 44). In this example, a beam-emitting apparatus orlight source 50 (e.g., an excitation source) illuminates the cartridge Cwith an excitation beam 51. At least a portion of the excitation beam istransmitted through the optical window 43 to the chamber 44 and resultsin an emission signal generated in the chamber 44. In this example, theemission beam 52 is directed toward a detector (not shown) located, forexample, at about 90°angle from the excitation beam. In other examples,alternative beam geometries can be used (e.g., with detection pathsdirected in various directions other than the source path 51, 53 and54). A portion of the excitation beam 51 can be scattered, absorbed orotherwise lost, as shown by the source path in FIG. 15. Upon reachingthe optical window 43, the excitation beam can be transmitted andabsorbed by the sample fluid in the chamber 44, absorbed as heat in thecartridge, and/or transmitted through the cartridge with being absorbed.The light that is not absorbed within the cartridge can exit as a beam54. A portion of the excitation beam 51 can be scattered or reflectedaway from the cartridge as beams 53 instead of being transmitted into orthrough the cartridge. Any description herein in relation to a (light)beam applies to light beams that are collimated, as well as light beamthat are not collimated, at least in some implementations. Further, anydescription herein in relation to a (light) beam applies to individuallight rays, and vice versa, at least in some implementations. Stillfurther, any description of optical elements herein in relation tooptical detection using the optical window 43, the chamber 44 and/or thecuvette 43, 44 applies to optical detection in one or more PCR chambersor elsewhere on the cartridge without the use of the optical window 43at least in some implementations. In some implementations, the opticalcomponents can be packaged to allow the cartridge to be inserted andremoved from a durable instrument without obstacles. In some cases, oneor more features (e.g., the optical components) can fit into a matingreceiving feature on the durable instrument to allow for improvedpositioning.

The source path can be affected and/or guided by optical components inthe optical window 43 and/or the cartridge C in order to improveutilization of the excitation light and enhance transmission of lightinto the chamber 44. Further, the source path can be affected and/orguided by optical components in the optical window 43 and/or thecartridge C in order to direct the excitation light 51, 53 and 54 awayfrom the detection direction 52. In some implementations, the opticalwindow 43 can comprise light guiding elements such as, for example,prisms, lenses, or Fresnel lenses. For example, the optical window cancomprise a prism 55 (e.g., a prism formed from a cyclo-olefin copolymeror other optically suitable material can be bonded to a top film of theoptical window 43 or to a top film of the cartridge C). In someimplementations, optical surfaces (e.g., surfaces facing the source pathor a portion of the source path) can include anti-reflective coatings tohelp transmit the excitation light to the interrogation volume or samplechannel (e.g., chamber 44, or one or more PCR chambers). In someimplementations, foils 56 and 57 (e.g., light-blocking foils) can beused on one or more surfaces (e.g., surfaces of the cartridge directedtoward the excitation light 51). Further, in some implementations,optical surfaces that allow scattered excitation light into thedetection path (e.g., detection path 52) can be coated or blocked usingfoil, paint or other structures. For example, black-painted surfaces 58can be provided on one or more interfaces of the optical plate oralignment fixture 48 (e.g., a cyclo-olefin copolymer or other opticallysuitable material can be bonded to a bottom (back) surface or film ofthe cartridge C) and/or on one or more interfaces of the prism 55.Light-directing features (e.g., lens or prism) and features to blockstray light from the excitation source (e.g., foil or coatings) can beused separately or in combination (e.g., synergistically combined).

FIG. 6 is an exploded view the ‘blister pack” disposable cartridge Cwith three layers comprising the layered sheets or membranes 31 and 32and the combined PSA layers 33, 34. The cartridge further comprises thechannels 7 and 8 for filling and extracting sample fluid sample from thechambers 4 and 5, respectively, and the channel 6 connecting thechambers 4 and 5. In some implementations, a pinch point (not shown) isprovided on the connecting channel 6. Such a pinch point can bereversible to allow for thermocycling of the sample fluid between thechambers 4 and 5.

FIG. 7 are cut-away and contour views of the assembled laminatedcartridge C with the optical window 43 embedded. Together with thechamber 44, the optical window can form the cuvette 43, 44. The cuvettemay or may not protrude from the cartridge surface as shown in FIG. 7.The cuvette, or any other optical detection configuration describedherein, can enable real-time PCR to be recorded. The cartridge furthercomprises the connecting channel 6 connecting the chambers 4 and 5, thefill/vent ports 45, the pinch point 46, and the channels 7 and 8 forfilling and extracting sample fluid sample from the chambers 4 and 5,respectively.

FIG. 8 shows a cartridge thermally modeled in Comsol assuming one-sidedor two-sided heating through polyimide membrane(s). The polyimidemembrane(s) can comprise one or more types of Kapton®, such as, forexample, Kapton® MT. The polyimide membrane(s) can be about 50 micron(50 μm) thick. The cartridge comprises the channels 6, 7 and 8,actuators, pinchers or pistons 47 for pinching/compressing the pinchpoints 46, plungers 10 and 11 for compressing the top walls of thechambers 4 and 5, respectively, a pair of heating/cooling elements 12,14 for heating top and bottom walls of the chamber 4, and a pair ofheating/cooling elements 13, 15 for heating top and bottom walls of thechamber 5. In this example, aluminum backed heating/cooling elements 12,14 and 13, 15 can be used. The chambers 4 and 5 can be formed fromlaminate layers. The top wall of each chamber can comprise the membrane31 (e.g., a Kapton® MT membrane) and the PSA layer 33. The bottom wallof each chamber can comprise the PSA layer 34 and the membrane 32 (e.g.,a Kapton® MT membrane). A heat conductive material, such as a 50 μm(0.002 inch) thick Kapton® MT interface, can be used between thechambers, and the heating/cooling elements 12, 14 and/or 13, 15. Whenone-sided heating is used, only one of the membranes 31 and 32 can beformed of a heat conductive material. For example, the top membrane 31can be formed of PDMS while the bottom membrane 32 can be formed ofKapton® MT. When two-sided heating is used, both membranes 31 and 32 canbe formed of a heat conductive material. For example, both membranes 31and 32 can be formed of Kapton® MT.

In an example, a nominal sample volume of 60 μL is contained withincartridge chambers with a total (combined) internal volume of about 60μL. Each of the chambers 4 and 5 can have an undeformed internal volumeof about 30 μL with a height (also “reduced sample volume height”herein) of about 250 μm. Upon actuation, one of the chambers (i.e., thechamber on the actuated side) is compressed and deformed (e.g., to afraction of its undeformed internal volume). The sample volume istransferred to the other chamber, which is expanded and deformed. Thedeformed internal volume of this chamber can be about 60 μL (e.g., sameas the sample volume). Upon subsequent actuation, the chamber that waspreviously expanded is compressed, the chamber that was previouslycompressed is expanded, and the situation is reversed. In some examples,the lower the height of the chamber, the larger the area that the samplevolume can be spread out over, leading to enhanced heat transfer rates.For example, large values of surface area to height or surface area tovolume ratios of the chambers can lead to increased heat transfer rates,and consequently decreased ramp times. In some examples, the height ofone or more of the chambers is less than about 500 μm, less than about400 μm, less than about 300 μm, less than about 250 μm, less than about200 μm, and the like.

FIGS. 9, 10, 11 and 12 provide thermal modeling results for thecartridge in FIG. 8. In FIGS. 9 and 11, fluid temperature in the coldchamber 4 immediately after fluid transfer from the hot chamber 5 at 95°C. is shown on the left, and a histogram of temperatures of the fluidentering the cold chamber 4 is shown on the right. In FIGS. 10 and 12,fluid temperature in the hot chamber 5 immediately after fluid transferfrom the cold chamber 4 at 55° C. is shown on the left, and a histogramof temperatures of the fluid entering the hot chamber 5 is shown on theright. The ambient temperature can be about 23° C. Two-sided heating inFIGS. 9 and 10 can result in reaching desired cartridge chambertemperatures within about 2 seconds. One-sided heating in FIGS. 11 and12 can result in reaching desired cartridge chamber temperatures withinabout 4 seconds.

FIG. 14 illustrates a recorded demonstration of PCR in a four-layerlaminated structure without a separate optical window, such as, forexample, the laminate structure in FIG. 3. The fluorescence can be readout through the low-temperature actuation button 37 through the PET toplayer 31. The cartridge can further comprise the laminate layer 31(e.g., comprising a 50.8 μm thick PET film), the laminate layer 33(e.g., comprising a 127 μm thick 3M 96042 Double-Coated SiliconeAdhesive layer), the laminate layer 34 (e.g., comprising a 127 μm thick3M 96042 Double-Coated Silicone Adhesive layer), and the laminate layer32 (e.g., comprising a 50.8 μm thick Kapton® MT film). In this example,the undeformed thickness (also “reduced sample volume height” herein) isabout 254 μm, and the 50 μL sample volume comprises about 21.25 μL of10⁵ DNA/mL B. Atro (i.e., total concentration of 4.25×10⁴ DNA/mL), 25 μLTaqMan® Master Mix, 1.25 μL of 10 ⁵ micromolar (μM) FAM probe, 1.25 μLof 36 μM B. Atro forward primer, and 1.25 μL of 36 μM B. Atro reverseprimer. An inflection point 49 measured fluorescence intensity as afunction of cycle number indicates successful PCR amplification of thesample. In this example, PCR temperatures are approximately 60° C. (333K) and approximately 95° C. (368 K).

Laminate layers (e.g., layered sheets or membranes, PSAs, etc.) of thedisclosure can have a given thickness. Laminate layers can havedifferent thicknesses. In some cases, one or more laminate layers canhave the same thickness. For example, PSA layers can have a firstthickness and membranes can have a second thickness. In some cases,laminate layers can have the same thicknesses. In some implementations,a subset of the laminate layers can a thickness based on requiredmechanical integrity. For example, laminate layers that are exposed toheating may need to exhibit a higher mechanical integrity (e.g., coupledto heat resistance) than laminate layers that are not exposed toheating. In another example, laminate layers that are exposed to ahigher degree of stretching and/or compression may need to exhibit ahigher mechanical integrity than laminate layers that experience lessstress (e.g., membranes may need to withstand a higher mechanical stressthan PSAs). Further, the thickness of each laminate layer can varyacross the area of the laminate layer. For example, the laminate layercan be thicker in an area that is exposed to a higher degree of stress(e.g., chamber pressure, tension, compression strength). In anotherexample, the laminate layer thickness can be reduced in areas whereoptical detection, heat transfer, pinching, filling, extraction, ventingand/or other cartridge manipulations are performed. As describedelsewhere herein, laminate layer thickness can further be used to definechamber volume. For example, the thickness of one or more PSA layers canbe larger than the thickness of one or more membranes in situationswhere thicker PSA layers are used to implement chambers with largerinternal volume(s). In another example, membrane thickness is adjustedto achieve a given mechanical integrity and/or heat transferperformance, while PSA thickness is adjusted to achieve a given internalvolume of chambers, or vice versa. Further, membrane thickness and/orPSA thickness can be adjusted to achieve a given mechanical integrity, agiven heat transfer performance, a given internal volume of chambers, orany combination thereof.

In some examples, a laminate layer, or a portion thereof, can have athickness of at least about 10 μm, at least about 20 μm, at least about30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm,at least about 70 μm, at least about 80 μm, at least about 90 μm, atleast about 100 μm, at least about 110 μm, at least about 120 μm, atleast about 130 μm, at least about 140 μm, at least about 150 μm, atleast about 160 μm, at least about 170 μm, at least about 180 μm, atleast about 190 μm, at least about 200 μm, and the like. In someexamples, a laminate layer of the disclosure can have a thickness of atless than about 10 μm, less than about 20 μm, less than about 30 μm,less than about 40 μm, less than about 50 μm, less than about 60 μm,less than about 70 μm, less than about 80 μm, less than about 90 μm,less than about 100 μm, less than about 110 μm, less than about 120 μm,less than about 130 μm, less than about 140 μm, less than about 150 μm,less than about 160 μm, less than about 170 μm, less than about 180 μm,less than about 190 μm, less than about 200 μm, and the like.

In some examples, a laminate layer has a thickness of about 10 μm, about15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm,about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about70 μm, about 75 μm, about 80 μm, about 90 μm, about 100 μm, about 110μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, or more. Inan example, a membrane formed of a heat conductive material can have athickness of about 30-70 μm or about 45-55 μm, while a PSA layer canhave a thickness of about 100-150 μm or about 125-130 μm.

Multichamber Cartridges

In one aspect, provided herein are multichamber cartridges for use in athermocycling reaction. In exemplary embodiments, a multichambercartridge is a component of a thermocycler or cartridge-based systemdescribed herein. In one embodiment, a multichamber cartridge comprisesa disposable portion of a thermocycler system. A thermocycler system,such as previously described herein accommodates one or moremultichamber cartridges for performing simultaneous thermocyclingreactions. In some embodiments, the multichamber cartridge comprises afirst chamber for holding a fluid at a first average temperature and asecond chamber for holding the fluid at a second average temperature.The second chamber is in fluid communication with the first chamber,wherein the fluid is transferred between the first chamber and thesecond chamber to achieve a transition from the first averagetemperature to substantially the second average temperature or viceversa. The first chamber and the second chamber are in fluid connectionvia a connecting channel. In an exemplary embodiment, a multichambercartridge is a disposable as described previously herein, for example, alaminated disposable cartridge, and vice versa. In another embodiment, acartridge or thermocycler as previously described further comprises oris operably connected to one or more components or elements of amultichamber cartridge as described below, for example, a buffersolution tube or blistered chamber.

An average temperature includes any temperature within 5° C. of the settemperature, for example, an average temperature for a reaction in a hotchamber set at 95° C. has a temperature from about 90° C. to about 100°C. In another embodiment, an average temperature includes anytemperature within 3° C. of the set temperature, for example, an averagetemperature for a reaction in a hot chamber set at 95° C. has atemperature from about 92° C. to about 98° C. In another embodiment, anaverage temperature includes any temperature within 2° C. of the settemperature, for example, an average temperature for a reaction in a hotchamber set at 95° C. has a temperature from about 93° C. to about 97°C. In another embodiment, an average temperature includes anytemperature within 1° C. of the set temperature, for example, an averagetemperature for a reaction in a hot chamber set at 95° C. has atemperature from about 94° C. to about 96° C. In certain instances, thetemperature of a heater fluctuates between average temperature valuesduring a thermocycling reaction. Alternatively, the temperature of aheater does not fluctuate temperature during a thermocycling reaction. Asubstantially average temperature includes a temperature within 5° C.,4° C., 3° C., 2° C., 1° C. or 0.5° C. of the average temperature. Theaverage temperature includes the temperature set in a thermocyclingreaction, the temperature of a heating element (e.g., heater asdescribed herein), the temperature of a fluid in chamber, and anycombination thereof.

In one embodiment, the multichamber cartridge comprises a third chamberfor holding a fluid at a third average temperature in fluidcommunication, directly or indirectly, with the first chamber, thesecond chamber, or both the first and second chambers. In anotherembodiment, a multichamber cartridge comprises a fourth chamber forholding a fluid at a fourth average temperature. In yet anotherembodiment, a multichamber cartridge comprises a fifth, sixth, seventh,eighth, ninth or tenth chamber for holding a fluid at a fifth, sixth,seventh, eighth, ninth or tenth average temperature, respectively. Invarious implementations, the third chamber, fourth chamber, fifthchamber, sixth chamber, seventh chamber, eighth chamber, ninth chamber,tenth chamber, or any combination thereof is an auxiliary chamber. Inone example, the auxiliary chamber is a blistered chamber. Inalternative implementations, the third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, or any combination thereof is not held at a fixedaverage temperature.

In one instance, the first chamber, second chamber, third chamber,fourth chamber, fifth chamber, sixth chamber, seventh chamber, eighthchamber, ninth chamber, tenth chamber, or any combination thereof isheld at an average temperature of between about 90° C. and about 110° C.for a given time during a thermocycling reaction. In one instance, thefirst chamber, second chamber, third chamber, fourth chamber, fifthchamber, sixth chamber, seventh chamber, eighth chamber, ninth chamber,tenth chamber, or any combination thereof is held at an averagetemperature suitable for denaturing nucleic acid molecules for a giventime during a thermocycling reaction. In one instance, the firstchamber, second chamber, third chamber, fourth chamber, fifth chamber,sixth chamber, seventh chamber, eighth chamber, ninth chamber, tenthchamber, or any combination thereof is held at an average temperature ofbetween about 40° C. and about 75° C. for a given time during athermocycling reaction. In one instance, the first chamber, secondchamber, third chamber, fourth chamber, fifth chamber, sixth chamber,seventh chamber, eighth chamber, ninth chamber, tenth chamber, or anycombination thereof is held at an average temperature suitable fornucleic acid annealing for a given time during a thermocycling reaction.In one instance, the first chamber, second chamber, third chamber,fourth chamber, fifth chamber, sixth chamber, seventh chamber, eighthchamber, ninth chamber, tenth chamber, or any combination thereof isheld at an average temperature of between about 60° C. and about 80° C.for a given time during a thermocycling reaction. In one instance, thefirst chamber, second chamber, third chamber, fourth chamber, fifthchamber, sixth chamber, seventh chamber, eighth chamber, ninth chamber,tenth chamber, or any combination thereof is held at an averagetemperature suitable for nucleic acid extension for a given time duringa thermocycling reaction. In one instance, the first chamber, secondchamber, third chamber, fourth chamber, fifth chamber, sixth chamber,seventh chamber, eighth chamber, ninth chamber, tenth chamber, or anycombination thereof is held at an average temperature suitable fornucleic acid digestion (e.g., with restriction enzymes) or ligation. Inone instance, the first chamber, second chamber, third chamber, fourthchamber, fifth chamber, sixth chamber, seventh chamber, eighth chamber,ninth chamber, tenth chamber, or any combination thereof is held at anaverage temperature suitable for temporarily storing or cooling nucleicacid molecules, for example from about 2° C. to about 25° C.

In one embodiment, a multichamber cartridge is configured to receive asample from a swab collection tube. In one implementation, the swabcollection tube is a removable component of a multichamber cartridge. Aswab collection tube retains a sample, optionally collected on a swab.For example, a sample is collected from a subject or environment andplaced in a sample collection tube for storage. The sample can then betransferred to a multichamber cartridge prior to a thermocyclingreaction. In one embodiment, the sample comprises nucleic acids to beamplified in an amplification reaction. In addition to a sample, theswab collection tube is configured to retain a swab solution, whichincludes reagents for preserving a sample, such as a biological sample,and includes, without limitation, buffer agents and salts. In anotherexample, a swab collection tube comprises processing reagents. In oneembodiment, the swab collection tube is fitted with a swab plunger fordelivering a sample solution to a chamber or other component of amultichamber cartridge. In one instance, the contents of a swabcollection tube, or a portion thereof, are delivered to a chamber of themultichamber cartridge via a receiving tube. In another instance, asample is processed in the swab collection tube. For example, a samplecomprising whole blood is processed to separate serum, wherein theprocessing reagents in the sample collection tube comprise a polymer geland a powdered glass clot activator.

In various aspects, a sample provided to a cartridge described hereincomprises one or more analytes suitable for a thermocycling reaction.Exemplary analytes include nucleic acid molecules to be amplified duringa thermocycling reaction. Samples include fluid and solid samples. Thesample includes any appropriate material, with any suitable origin. Forexample, a sample includes, without limitation, a biomolecule,organelle, virus, cell, tissue, organ, and/or organism. A sampleoptionally is a biological sample, such as blood, urine, saliva, sweat,seminal fluid, tissue, amniotic fluid, cerebrospinal fluid, synovialfluid, tears, fecal matter, and/or mucous, among others. A sampleoptionally is an environmental sample, such as a sample from air, water,or soil. In one embodiment, a sample is aqueous and optionally comprisesbuffering agents, inorganic salts, and/or other components known forassay solutions. Suitable samples include compounds, mixtures, surfaces,solutions, emulsions, suspensions, cell cultures, fermentation cultures,cells, tissues, secretions, and/or derivatives and/or extracts thereof.A sample includes food products. In some instances, a sample is providedto the cartridge in a processed form altered from its original state.For example, it may be necessary to lyse or permeabilize cells torelease nucleic acids. Such methods include chemical (e.g., Lysozyme),mechanical (e.g., sonication), thermal, or a combination thereof. Asanother example, a sample comprising a pathogenic organism is chemicallyor thermally inactivated. In some instances, analytes (e.g., nucleicacids) of a processed sample are isolated or separated in one or moretubes or chambers of the cartridge.

In one embodiment, a multichamber cartridge comprises a receiving tubefor receiving a sample. In one implementation, the sample is provided inwhole or in part from a swab collection tube. In another example, asample (optionally processed) is provided to the receiving tube directlyvia an opening, for example, a fill port, by any means suitable fortransferring a liquid or semi-liquid solution, e.g., syringe, pipette orplunger. In one embodiment, the receiving tube is a disposable and/orremovable component of a thermocycler.

In one embodiment, a multichamber cartridge comprises one or more buffersolution tubes, wherein each buffer solution tube is configured to holdone or more buffers or aqueous solutions. In one embodiment, amultichamber cartridge comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morebuffer solution tubes. In an exemplary embodiment, a multichambercartridge comprises 3 buffer solution tubes. In one instance, themultichamber cartridge is formulated to hold one or more buffers in theone or more buffer solution tubes, for example, the multichambercartridge is manufactured with buffer solution(s) stored in the tube(s).In one embodiment, the buffers are supplied in a cartridge as aqueoussolutions or as dehydrated pellets to be reconstituted with an aqueoussolution. In another instance, the multichamber cartridge is suppliedwith buffers by a thermocycler end-user, wherein the buffer componentsare optimized for a specific thermocycling reaction. A buffer or aqueoussolution comprise any components useful for carrying out a thermocyclingreaction, e.g., a nucleic acid amplification reaction. Suitablecomponents include, without limitation, primers, probes, polymerases,nucleotides, divalent cations, and monovalent cations. In oneembodiment, a buffer solution tube holds volumes up to about 5 mL, up toabout 4 mL, up to about 3 mL, up to about 2 mL, up to about 1 mL, up toabout 0.5 mL, up to about 0.4 mL, up to about 0.3 mL, up to about 0.2 mLor up to about 0.1 mL. In one embodiment, one or more buffer solutiontubes are disposable and/or removable components of a thermocycler.

In one embodiment, a multichamber cartridge comprises a means fordelivering a buffer solution to a chamber of the cartridge. For example,a buffer solution is delivered to a chamber of the cartridge byapplication of a plunger. In the instances wherein a multichambercartridge comprises a plurality of buffer solution tubes, one or morebuffer solutions are delivered to one or more chambers via a rotatingplunger. In one embodiment, the plunger is a disposable and/or removablecomponent of a thermocycler.

In one embodiment, a multichamber cartridge comprises a rotating filtermembrane comprising a plurality of channels in connection with one ormore tubes or chambers of the cartridge, wherein the plurality ofchannels are opened or closed by rotating the rotating filter membrane.In one embodiment, the rotating filter membrane is in fluid connectionwith a receiving tube. In one embodiment, the rotating filter membraneis in fluid connection with one or more buffer solution tubes. In oneembodiment, the rotating filter membrane is in fluid connection with awaste chamber. In another embodiment, the rotating filter member is influid connection with a blistered chamber. In another embodiment, therotating filter membrane is in fluid connection with the first chamberor cold chamber. In yet another embodiment, the rotating filter membraneis in fluid connection with the second or hot chamber. In furtherembodiments, the rotating filter membrane is in fluid connection with athird, fourth, fifth, sixth, seventh, eighth, ninth, tenth chamber orany combination thereof. In one embodiment, the rotating filter membraneis a disposable and/or removable component of a thermocycler.

In one embodiment, a multichamber cartridge comprises a waste chamber oralternatively, a waste tube. The waste chamber holds waste from one ormore tubes and/or one or more chambers of a multichamber cartridge. Insome embodiments, the waste chamber holds volumes up to about 10 mL, upto about 8 mL, up to about 7 mL, up to about 6 mL, up to about 5 mL, upto about 4 mL, up to about 3 mL, up to about 2 mL, up to about 1 mL, orup to about 0.5 mL. In one embodiment, the waste chamber is a disposableand/or removable component of a thermocycler.

In one embodiment, a multichamber cartridge comprises a blisteredchamber, for example, a blistered chamber as previously described. Inone embodiment, the blistered chamber is in fluid connection with arotating filter membrane. In another embodiment, the blistered chamberis in fluid connection with a first or cold chamber. In anotherembodiment, the blistered chamber is in fluid connection with a secondor hot chamber. In another embodiment, the blistered chamber is in fluidconnection with a third, fourth, fifth, sixth, seventh, eighth, ninth,tenth chamber or any combination thereof. In some implementation, theblistered chamber is compressed by an actuator to move contents to otherchambers of the cartridge, for example, the first or second chambers. Inone instance, the actuator is a component of a thermocycler providedherein. In one embodiment, the blistered chamber is a disposable and/orremovable component of a thermocycler. The blistered chamber, in manyinstances, comprises reagents useful for sample preparation and/orperforming a thermocycling reaction. Such reagents are manufactured withthe cartridge or added by an end-user prior to performing athermocycling reaction. Suitable reagents include, without limitation,Lysozyme and Proteinase K. In one embodiment, the reagents areformulated as a solution or a powder. In one embodiment, one or morereagents of the blistered chamber are activated by a heater in a heatactivation step. In an additional embodiment, the blister chamber is incontact with a heater.

In an exemplary embodiment, a fluid comprising a sample is moved fromone chamber to another, e.g., between a first and second chamber, bydeforming one chamber with pressure from one or more actuators aspreviously described.

In one embodiment, a multichamber cartridge comprises a window orcuvette to view a fluid in the cartridge. In an exemplary embodiment,the window is an optical viewing window useful for fluorescentdetection. The optical viewing window is comprised of opticallytransparent material for receiving and transmitting light.

The chambers of the multichamber cartridge are of any size suitable fortheir respective functions in an amplification reaction. For example,any chamber of the multichamber cartridge holds a volume of solution(e.g., sample reaction solution, buffer solution, waste solution,blistered chamber solution) from about 5 μL to about 5 mL. Similarly,the tubes of the multichamber cartridge are of any size to accommodatevolumes from about 5 μL to about 5 mL. In one embodiment, one or moretubes, chambers and/or other multichamber components are operablyconnected to or attached to the multichamber cartridge. In variousimplementations, any number of components, e.g., tube, chamber, areremovable or non-removable components.

In one embodiment, one or more chambers or components of a cartridgeprovided herein is adapted for nucleic acid extraction, nucleic acidpurification, nucleic acid detection and/or nucleic acid amplification.

In one embodiment, the multichamber cartridge is a disposable portion ofa thermocycler. In another embodiment, the multichamber cartridge isuseful with a thermocycler described herein. In another embodiment, themultichamber cartridge is a laminated disposable cartridge.

In one embodiment, the multichamber cartridge comprises plastic injectedpolycarbonate material. In one embodiment, the multichamber cartridgecomprises Kapton laminate. In another embodiment, the multichambercartridge comprises PSA. In a further embodiment, the multichambercartridge comprises PET laminate. Additional multichamber cartridgematerials include, without limitation, cardboard, plastic and paper.

In one embodiment, one or more components of the multichamber cartridgeare labeled. For example, a label provides information that is readableto a human or machine to provide information relating to a sample. Alabel includes a scannable barcode.

In one aspect, fluid is transferred between one or more chambers of acartridge provided herein and one or more chambers and/or cartridgecomponents provided herein, by mechanical actuation. In otherembodiments, fluid is transferred without using movable components of acartridge and/or thermocycler. In some embodiments, fluid is transferredusing magnetic means, for example, by using magnetic fluids or magneticbeads.

In various implementations, one or more chambers of the cartridgesdescribed herein are pre-loaded with reagents. Reagents include liquids,solids, gases or combinations thereof. In an additional embodiment, oneor more chambers of the cartridge comprises at least one opening toallow for the addition or removal of a sample. In one embodiment, theopening is sealed or otherwise closed to maintain the sample in thechamber. In another or additional embodiments, a chamber is initiallyempty so as to serve, for example, as a waste, mixing and/or detectionchamber prior to, during or after a thermocycling reaction. Reagentsinclude those which are formulated as a pellet or tablet, or arelyophilized. In an example, reagents are useful for sample preparation(e.g., immobilization), amplification (e.g., primers), or detection(e.g., probes). In one embodiment, a solid reagent is re-suspended witha fluid, for example, one provided by an adjacent chamber or tube. In anadditional embodiment, a chamber comprises cryoprotectants or preservingagents such as disaccharides (e.g., trehalose, sucrose).

In various implementation, one or more chambers of the cartridge areuseful for processing amplified nucleic acids after completion ofthermocycling. For example, a chamber comprises one or more reagents,either pre-loaded or provided by an end-user, for performing arestriction digest on amplified nucleic acids. In another or additionalembodiment, a chamber comprises one or more reagents, either pre-loadedor provided by an end-user, for performing a ligation reaction.

A cartridge is loaded with a sample by manual, automated, or manual andautomated methods. Sample loading methods include, without limitation,pipetting, injection, spotting, and syringe drawing.

An exemplary multichamber cartridge is provided in FIG. 16. Themultichamber cartridge in this embodiment comprises a first chamber orcold chamber 104 for holding a fluid at a first average temperature anda second chamber or hot chamber 105 for holding a fluid at a secondaverage temperature. The first chamber 104 and the second chamber 105 isin fluid connection via a connecting channel 106. The connecting channel106 comprises a viewing window 143 for fluid detection. In oneembodiment, the window is an optical viewing window for fluorescentdetection.

The multichamber cartridge of FIG. 16 further comprises a swabcollection tube 161 for holding a sample 160, wherein the swabcollection tube optionally comprises a plunger 162. The sample, andoptionally a buffer, is transferred to a receiving tube 163 via thedepression of the plunger 162.

The multichamber cartridge of FIG. 16 further comprises three buffersolution tubes, 164 a, 164 b and 164 c. In one embodiment, the buffersolution tubes have a volume from about 10 μL to about 2 mL. In oneembodiment, buffer solution tube 164 a has a volume of about 200 μL,buffer solution tube 164 b has a volume of about 200 μL and buffersolution tube 164 c has a volume of about 300 μL. The buffer solutionsare transferred to a rotating filter membrane 166 by a rotating plunger165. The rotating filter membrane comprises a plurality of channels inconnection with the receiving tube 163, at least one buffer solutiontube 164, a waste chamber 167, and a blistered chamber 168, wherein theplurality of channels are opened or closed by rotating the rotatingfilter membrane. The waste chamber 167 functions to collect wastesolution from other components of the cartridge. The cartridge embodiedin FIG. 16 further comprises a blistered chamber 168. In one example,the blistered chamber is 12 mm in diameter and 0.5 mm high. In oneembodiment, one or more reagents are stored in this chamber, for exampleLysozyme and/or Proteinase K.

In one embodiment, a rotating filter membrane 166 controls release of asample from the receiving tube 163 to a blister chamber 168 and/or awaste chamber 167. In one embodiment, a rotating filter membrane 166controls release of a buffer from one or more buffer tubes 165 to ablister chamber 168 and/or a waste chamber 167.

Cartridge-Based Thermocycler Methods

In one aspect, provided herein are cartridge-based thermocyclers andthermocycling systems comprising one or more cartridges. In oneembodiment, a thermocycler described herein is configured to perform athermocycling reaction with at least two cartridges at the same time. Inone example, one cartridge comprises a reference sample and anothercartridge comprises a test sample. For instance, in a diagnostic assay,a reference or control sample is amplified under the same conditions asa test sample.

The cartridge-based thermocyclers provided herein allow for thequantitative or qualitative detection of a target nucleic acid in asample. For example, during a nucleic acid amplification reaction, atarget component in a sample is detected when one or more detectablylabeled probes hybridize to the target and to the amplification productsthereof. In many instances, the detection of the target is indicative ofa disease presence in the sample, for example, when the target is anucleic acid from an infectious agent. In another instance, theexpression level of a target nucleic acid in a sample is quantifiedduring a PCR reaction. In some instances, expression level is indicativeof a disease state or a correlation to a disease state. For example,differential expression of a nucleic acid expression product, e.g., RNA,as compared to a reference expression level, is indicative of a diseasestate.

In another aspect, provided herein are thermocycling systems useful forforensic applications, for example, in genetic fingerprinting.

In another aspect, provided herein are thermocycling systems useful forDNA sequencing.

In exemplary embodiments, the thermocycling systems provided herein canbe employed to detect and/or quantify two or more different nucleicacids in a sample. For example, the nucleic acids can be quantifiedaccording to the rate at which they can be amplified detectably from thesample by an amplification reaction in which the nucleic acids arecopied exponentially and/or linearly. Any suitable amplificationapproach can be used, including PCR. In some embodiments, probes fordifferent nucleic acid targets in a sample are labeled with a differentdetectable label (e.g., fluorescent label). Hybridization of a probe tothe target can produce a change in light emission from the detectablelabel. A suitable assay that can quantify nucleic acids according to therate of change in light emission is exemplified by a TaqMan® assay(Applied Biosystems). The cartridges, thermocyclers, and systemsprovided herein are useful for such thermocycling reactions.Thermocycling reactions include nucleic acid amplification reactions.Such reactions include, without limitation, PCR, real-time PCR,allele-specific PCR, SNP genotyping, assembly PCR (e.g. nucleic acidsynthesis), asymmetric PCR, helicase-dependent amplification, ligationmediated PCR, quantitative PCR, and reverse transcription PCR. In oneembodiment, the cartridges, thermocyclers and systems provided hereinare used for multiplex-PCR.

The detection and/or measurement of amplification products are performedat reaction completion or in real time (i.e., during reaction), wherereal time includes continuous or discontinuous measurement and/ordetection. If the measurement of accumulated amplified product isperformed after amplification is complete, the labeled probes can beadded after the amplification reaction. Alternatively, probes are addedto the reaction prior to or during the amplification reaction.

In another aspect, provided herein are cartridge-based thermocyclers forperforming real-time PCR. Real-time PCR, in various implementations, isuseful to simultaneously amplify and detect or quantify a nucleic acidmolecule in a sample. In one embodiment, amplified nucleic acidmolecules are detected using detectable dyes, e.g., fluorescent dyes,that intercalate with DNA. In another embodiment, amplified nucleic acidmolecules are detected using nucleic acid probes comprising detectablelabels. In one embodiment, a probe comprises a plurality of detectablelabels. Detectable labels are naturally and/or artificially occurring.Naturally occurring labels include green fluorescent protein (GFP),phycobiliproteins, luciferase, and/or their many variations, amongothers. Artificially occurring labels include, for example, rhodamine,fluorescein, FAM™/SYBR® Green I, VICO/JOE, NED™/TAMRA™/Cy3™, ROX™/TexasRed®, Cy5™, among others. Suitable natural and artificial labels aredisclosed in the following publication, among others, which isincorporated herein by reference: Richard P. Haugland, Handbook ofFluorescent Probes and Research Chemicals (6th ed. 1996). In someembodiments, a fluorescently labeled probe is active only in thepresence of a target molecule, for example, a specific nucleic acidsequence, so that a fluorescent response from a sample signifies thepresence of the target molecule. In exemplary embodiments, a probe is ahybridization probe comprising an oligonucleotide which hybridizes to atarget nucleic acid sequence that is complementary to theoligonucleotide probe sequence.

In some embodiments, a probe is a molecular beacon. A molecular beaconprobe, as described herein, includes a single-stranded oligonucleotidein which the bases on the 3′ and 5′ ends are complementary, forming astem. A molecular beacon probe forms a hairpin structure at temperaturesat and below those used to anneal the oligonucleotide to a target. Insome embodiments, the molecular beach probe forms a hairpin structure attemperatures below about 60° C. The double-helical stem of the hairpinbrings a fluorophore (or other label) attached to the 5′ end of theprobe in proximity to a quencher attached to the 3′ end of the probe.The probe does not fluoresce (or otherwise provide a signal) in thisconformation. If a probe is heated above the temperature needed to meltthe double stranded stem apart, or the probe hybridizes to a targetnucleic acid that is complementary to the sequence within thesingle-strand loop of the probe, the fluorophore and the quencher areseparated, and the fluorophore fluoresces in the resulting conformation.Therefore, in a series of nucleic acid amplification cycles the strengthof the fluorescent signal increases in proportion to the amount of themolecular beacon that is hybridized to the target, when the signal isread at the annealing temperature. Molecular beacons of highspecificity, having different loop sequences and conjugated to differentfluorophores, can be selected in order to monitor increases in ampliconsthat differ by as little as one base.

During a real-time PCR reaction, fluorescence intensity is monitored inreal time. A key element in the measurement is to identify the thermalcycle number at which the label emission intensities rise abovebackground noise and starts to increase, preferably exponentially. Thiscycle number is called the threshold cycle, C₁. The C₁ value isinversely proportional to the number of starting copies of the DNAsample in the original PCR solution. Knowing C₁, the quantity of the DNAto be detected in the sample can be determined.

In one aspect, provided herein are thermocycling systems useful for theidentification and management of a disease state. For example, DNAfragments comprising a gene or expression product thereof can beamplified and detected using one or more detectably labeled probes aspreviously described. In one embodiment, a detectably labeled probecomprising a sequence indicative of a disease state comprises a distinctfluorophore which is detected using an optical detector. For example, asequence indicative of a disease state comprises one or more mutations.In some embodiments, the presence of a sequence of nucleic acid isidentified using a thermocycling system described herein. In oneexample, a nucleic acid sequence from an infectious disease agent (e.g.,bacteria, virus) is present in a human or environmental sample. Thenucleic acid sequence is amplified and detected by hybridization with adetectably labeled probe. In some embodiments, a plurality of dectablylabeled probes are provided in an amplification reaction, wherein uniquecombinations of probes are indicative of a disease state, allowing for amultiplex reaction to be performed within one cartridge. In otherembodiments, a plurality of cartridges are used in a thermocyclingsystem provided herein.

In another aspect, provided herein is a method for monitoring athermocycling reaction, the method comprising a) providing athermocycler comprising a first chamber for holding fluid at a firstaverage temperature and a second chamber for holding the fluid at asecond average temperature, wherein the second chamber is in fluidcommunication with the first chamber, b) introducing a sample intoeither the first chamber or the second chamber, wherein the samplecomprises a nucleic acid molecule and one or more detectably labeledprobes configured to hybridize to the nucleic acid molecule; c)transferring the sample from the first chamber to the second chamber;and d) measuring a detectable signal emitting from the sample inresponse to a stimulus using an optical detector. In some embodiments,the detectable signal comprises both a signal correlating to nucleicacid amplification and a signal correlating to noise. In some instances,the signal correlating to nucleic acid amplification is indicative of aquantity of amplified nucleic acid in the sample. In some embodiments,the signal correlating to nucleic acid amplification is distinguishablefrom the signal correlating to noise.

EXAMPLES Example 1: Thermocycler Cartridge

A cartridge of a cartridge-based thermocycler comprises a four-layeredlaminated structure having two chambers in fluid connection via aconnecting channel. One or both of the chambers is connected to one ormore addition channels to allow for filling a chamber with fluid orextracting fluid from a chamber. The structure is manufactured with orwithout an optical window. If the structure is manufactured without theoptical window, an optical detection signal can be read through a toplayer of the structure, preferably over a chamber of the cartridge. Thecartridge comprises a laminate layer having a 50.8 μm thick PET film, alaminate layer having a 127 μm thick 3M 96042 Double-Coated SiliconeAdhesive layer, another laminate layer having a 127 μm thick 3M 96042Double-Coated Silicone Adhesive layer, and a laminate layer having a50.8 μm thick Kapton® MT film. The thickness of each chamber is 254 μm.A working example of this cartridge is shown in FIG. 14A.

Example 2: Method of Nucleic Acid Amplification Using a Cartridge-BasedThermocycler

A cartridge as described in Example 1 is part of a thermocycling systemcomprising a thermocycling instrument. The thermocycling instrumentcomprises two heater blocks, wherein one heater block provides heat at afirst average temperature to the first chamber and the second heaterblock provides heat at a second average temperature to the secondchamber. The thermocycling instrument comprises two actuators, whereinone actuator provides pressure to one chamber and the second actuatorprovides pressure to the second chamber, and wherein said pressure issufficient to propel a fluid from one chamber into another chamber.

A 50 μL nucleic acid amplification reaction was prepared comprising21.25 μL of 10⁵ DNA/mL B. Atro, 25 μL TaqMan® Master Mix, 1.25 μL of 105μM FAM probe, 1.25 μL of 36 μM B. Atro forward primer and 1.25 μL of 36μM B. Atro reverse primer. The reaction mixture was added to a firstchamber of the cartridge through a channel. The cartridge was placed ina slot of the thermocycling instrument and a thermocycling program wasset. Thermocycling conditions were: 95° C. for 30 seconds; 55 cycles of95° C. for 10 seconds and 60° C. for 10 seconds. During thethermocycling reaction, actuators of the instrument applied pressurealternatively to each chamber, propelling the reaction mixture from onechamber to another through a connecting channel, while each chamber wasmaintained at an average temperature by each heater. One chamber wasmaintained at an average temperature of 60° C. and the other chamber wasmaintained at an average temperature of 95° C.

The reaction was monitored in real-time by measuring fluorescenceintensity as a function of cycle number. The fluorescence intensity datais shown in FIG. 14B. The C₁ value was 28.4 for this reaction. The timeto reach 30 cycles was 10 minutes, 30 seconds (10 minutes of cycling anda 30 second hot start).

Example 3: Molecular Diagnosis of a Disease Using Real-Time PCRPerformed With a Cartridge-Based Thermocycler

This example describes a quantitative real-time RT-PCR assay for thedetection of an infectious disease from a patient sample, in thisinstance serum. This assay targets a nucleic acid region specific insequence for the disease, in this case a virus, and utilizesfluorescently labeled probes for detection. This assay is similarlyperformed utilizing a plurality of probes targeting additionalinfectious diseases in a multiplexed assay. Alternatively, this assay isperformed for serotyping a disease by utilizing a plurality of probestargeting different serotypes of the disease. These optional multiplexedassays are performed in a single cartridge of a thermocycler describedherein.

The real-time RT-PCR reaction is performed using a thermocyclerdescribed in Examples 1 and 2, which is operably attached to an opticaldetector, and a commercial qRT-PCR kit (SuperScript One-Step qRT-PCR,Life Technologies). The connecting channel of the cartridge has anoptical window to allow for excitation light from the optical detectorto reach the sample and to allow for emission light from the reactionmixture to reach the detector. Thus, during the thermocycling reaction,as the detectably labeled probe hybridizes to amplification products,the optical detector records fluorescence signals in real-time.

The real-time RT-PCR reaction mixture comprises a forward primer andreverse primer designed to amplify a target nucleic acid region of thevirus. Nucleic acids are extracted from serum samples of a patient usingthe QIAamp Viral RNA Mini Kit (Qiagen) using recommended procedures. Theextracted nucleic acids are added to the reaction mixture atconcentrations between about 1 pg to about 1 μg total RNA. A TaqMan®probe is added to the final PCR reaction. The probe is added to thereaction via a blistered chamber or directly to the reaction mixture.

Thermocycling conditions are: 50° C. for 15 minutes (RT step); 95° C.for 2 minutes; 40 cycles of 95° C. for 2 seconds and 60° C. for 2seconds. The total cycle time is less than about 25 minutes and the PCRtime is less than about 10 minutes.

The reaction is monitored in real-time using an optical detector todetect fluorescence of the detectably labeled probe hybridized toamplified target nucleic acids.

A positive diagnosis of the infectious disease is considered when asample has a C₁ value less than about 35.

This diagnosis is performed as a point-of-care assay. As an example, thesample is whole blood which is processed in a sample processingcartridge prior to addition to the thermocycling cartridge.Alternatively, the sample is processed in one or more chambers of athermocycling cartridge as a first step in a thermocycling reaction.

Example 4: Cartridge Kit

A disposable portion of a thermocycler is provided in the form of asingle cartridge. The cartridge comprises at least two chambersconnected by a channel to allow fluid to flow between the chambers. Thechambers are constructed from suitable material to allow forcompression. One or both chambers is connected, e.g., through a channel,to one or more ports, to allow for the addition or removal of a liquid,gas, solid or combination thereof. The cartridge is customizable withadditional elements.

One additional element is a means to allow for the optical detection ofa substance within the cartridge. This means includes an optical windowlocated in a chamber or connecting channel. Another means is a cuvetteconnected by a fluid path to one or more chambers. The cartridge isconfigured for optical detection in real-time or at one or more timepoints during use.

Another additional element is a blistered chamber. The blistered chamberis similarly customizable to comprise reagents for use in athermocycling reaction. Alternatively, the blistered chamber is suppliedempty, allowing for an end-user to add reagents.

The cartridge is optionally provided with an instrument in athermocycler system. The instrument is able to act on all or a portionof the cartridge and such actions are programmable. Such actions includemoving a fluid between chambers, opening and closing channels betweenchambers, providing a barrier between chambers, heating or cooling achamber, and any combination thereof. The instrument is configured to beprovided with, or configured to be operably connected to, an opticaldetector.

It is to be understood that the terminology used herein is used for thepurpose of describing specific embodiments, and is not intended to limitthe scope of the present invention. It should be noted that as usedherein, the singular forms of “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. In addition,unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

While preferable embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A molecular diagnostic test apparatus, comprising: a fluid chamberformed by a top wall and a bottom wall, the top wall including a curvedsurface and the bottom wall including a curved surface such that thefluid chamber is shaped to decrease bubbles being trapped when a sampleflows into the fluid chamber; the bottom wall comprising an opticalmaterial configured to transmit an optical signal into and out of thefluid chamber; a heater disposed adjacent the bottom wall, the heaterconfigured to increase a temperature of the sample within the fluidchamber to amplify a target nucleic acid within the sample; a lightsource configured to produce the optical signal, the optical signalbeing transmitted through the optical material of the bottom wall andinto the fluid chamber; and an optical detector in optical communicationwith the fluid chamber via the optical material of the bottom wall, theoptical detector configured to receive a detected signal associated witha presence of the target nucleic acid within the sample.
 2. Themolecular diagnostic test apparatus of claim 1, wherein the fluidchamber has a tear drop shape.
 3. The molecular diagnostic testapparatus of claim 1, wherein the curved surface of the fluid chamberhas a curvature that is larger than a curvature of a wavefront of thesample as the sample flows through the fluid chamber.
 4. The moleculardiagnostic test apparatus of claim 1, wherein the fluid chamber is afirst fluid chamber, the curved surface of the top wall is a firstcurved surface of the top wall, the curved surface of the bottom wall isa first curved surface of the bottom wall, the apparatus furthercomprising: a second fluid chamber formed by the top wall and the bottomwall, the top wall including a second curved surface and the bottom wallincluding a second curved surface such that the second fluid chamber isshaped to decrease bubbles being trapped when a sample flows into thesecond fluid chamber.
 5. The molecular diagnostic test apparatus ofclaim 1, wherein the light source produces a light beam at the opticalmaterial of the bottom wall; and the optical detector receives thedetected signal associated with the presence of the target nucleic acidwithin the sample at a transverse angle relative to the light beam. 6.The molecular diagnostic test apparatus of claim 1, wherein the lightsource produces a light beam at the optical material of the bottom wall,the optical detector receives the detected signal associated with apresence of a target nucleic acid within the sample at about a 90 degreeangle relative to the light beam.
 7. The molecular diagnostic testapparatus of claim 1, further comprising: an optical component toenhance transmission of light from the light source into the fluidchamber.
 8. The molecular diagnostic test apparatus of claim 7, whereinthe optical component includes at least one of a lens, ananti-reflective component, or a light blocking component.
 9. Themolecular diagnostic test apparatus of claim 1, further comprising: asample receiving port in fluid communication with the fluid chamber; anda sample transfer device having an end portion configured to couple tothe sample receiving port to convey the sample from within the sampletransfer device into the fluid chamber.
 10. The molecular diagnostictest apparatus of claim 1, wherein the heater is disposed beneath thebottom wall.
 11. A molecular diagnostic test apparatus, comprising: acartridge defining a first fluid chamber and a second fluid chamber, thefirst fluid chamber and the second fluid chamber each being shaped todecrease bubble formation within the first fluid chamber and the secondfluid chamber when a sample flows into the first fluid chamber and thesecond fluid chamber; a sample receiving port defined within thecartridge; and a sample transfer device having an end portion configuredto couple to the sample receiving port to convey the sample from withinthe sample transfer device into the cartridge.
 12. The moleculardiagnostic test apparatus of claim 11, wherein the first fluid chamberand the second fluid chamber are formed by a top structure and a bottomstructure, the top structure including a first curved surface associatedwith the first fluid chamber and a second curved surface associated withthe second fluid chamber, the bottom structure including a first curvedsurface associated with the first fluid chamber and a second curvedsurface associated with the second fluid chamber.
 13. The moleculardiagnostic test apparatus of claim 12, wherein: the bottom structureincludes an optical material configured to transmit an optical signalinto and out of at least one of the first fluid chamber or the secondfluid chamber, the apparatus further comprising: a light sourceconfigured to produce the optical signal, the optical signal beingtransmitted through the optical material of the bottom structure andinto the at least one of the first fluid chamber or the second fluidchamber.
 14. The molecular diagnostic test apparatus of claim 13,further comprising: an optical detector in optical communication withthe at least one of the first fluid chamber or the second fluid chambervia the optical material of the bottom structure, the optical detectorconfigured to receive a detected signal associated with a presence of atarget nucleic acid within the sample.
 15. The molecular diagnostic testapparatus of claim 12, further comprising: a heater configured tocontact at least one of the top structure or the bottom structure, theheater configured to increase a temperature of the sample within atleast one of the first fluid chamber and the second fluid chamber toamplify a target nucleic acid within the sample.
 16. The moleculardiagnostic test apparatus of claim 14, wherein the light source producesa light beam at the optical material of the bottom structure, theoptical detector receives the detected signal associated with a presenceof a target nucleic acid within the sample at a transverse anglerelative to the light beam.
 17. The molecular diagnostic test apparatusof claim 14, wherein the light source produces a light beam at theoptical material of the bottom structure, the optical detector receivesthe detected signal associated with a presence of a target nucleic acidwithin the sample at about a 90 degree angle relative to the light beam.18. The molecular diagnostic test apparatus of claim 14, furthercomprising: an optical component within the cartridge to enhancetransmission of light from the light source into the at least one of thefirst fluid chamber or the second fluid chamber.
 19. The moleculardiagnostic test apparatus of claim 18, wherein the optical componentincludes at least one of a lens, an anti-reflective component, or alight blocking component.
 20. The molecular diagnostic test apparatus ofclaim 11, wherein the first fluid chamber and the second fluid chambereach have a tear drop shape.
 21. The molecular diagnostic test apparatusof claim 11, wherein the first fluid chamber and the second fluidchamber are fluidically coupled via a channel.